Rh blood group antigen compositions and methods of use

ABSTRACT

Disclosed are novel protein and peptide compositions comprising soluble and bound forms of immunologically-active blood group antigens including mammalian Rh antigens. In preferred embodiments methods for the isolation and purification of serologically-active human Rh antigens such as D, c, C, E, and e are disclosed. Also disclosed are methods for the adsorption of immunologically-active Rh antigens to solid supports. Diagnostic kits, methods, and devices for the detection of Rh antibodies in clinical and non-clinical samples are also disclosed. Devices, compositions and methods for the isolation, purification and quantitation of anti-Rh antibodies from solution are also provided.

This application is a divisional of co-pending application Ser. No.09/164,789, filed Oct. 1, 1998, now U.S. Pat. No. 6,191,108, which is adivisional of application Ser. No. 08/715,173, filed Sep. 17, 1996 nowU.S. Pat. No. 5,840,585.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates to the fields of protein chemistry andhematology. More particularly, the invention discloses novelcompositions comprising solid-phase, ie., bound, forms ofimmunologically-active Rh antigen. Also disclosed are diagnostic kitsand devices for the detection and quantitation of Rh antibodies inclinical and non-clinical samples. In another aspect, the inventionrelates to devices, compositions and methods for the isolation,identification, quantitation, and purification of anti-Rh antibodiesfrom solution.

1.2 Description of the Related Art

1.2.1 Rh Antigens

The Rh blood group system is one of the most complex polymorphisms inhumans. Human red blood cells (RBCs) may be subdivided into Rh⁺ and Rh⁻groups according to the presence or absence of the major Rh blood groupantigen, Rhesus D (Rh_(o) D) (Cartron and Agre, 1993). Several geneshave been implicated as encoding the major Rh antigen epitopes, D, C, c,E, and e, while a host of others are speculated to be involved in thedeterminants of a host of rare alleles.

Rh antigens, including Rh_(o) D, are carried on an integral membraneprotein which has a molecular weight of approximately 30 kDa (Moore etal., 1982; Gahmberg, 1982; 1983). This protein has been implicated inthe molecular adhesion of the submembranous cytoskeleton to theerythrocyte cell membrane (Ridgwell et al., 1984), and persons lackingthe proteins exhibit Rh Deficiency Syndrome, accompanied by varyingdegrees of hemolytic anemia (Marsh, 1983).

Paradis et al. (1986) demonstrated that the presence of the cytoskeletonin isolated Rh_(o) D antigen preparations served as a protective effecton the immunologic activity of the Rh antigen.

1.2.2 Hemolytic Disease of the Newborn (HDN)

The RBC antigen system in humans is the basis for the disease calledHemolytic Disease of the Fetus/Newborn. This disorder is manifested whenan Rh⁻ woman becomes pregnant by an Rh⁺ man. The fetus is statisticallylikely to be Rh⁺ and during gestation or at birth, Rh⁺ fetal RBC canenter the maternal circulation and the woman then has a high probabilityof developing an anti-Rh antibody response against the transferred RBC.In subsequent pregnancies, the IgG form of the antibody crosses theplacenta and enters the fetal circulation where it binds to fetal Rh⁺RBC and thereby causes them to be rapidly removed from circulation inliver and spleen. The first child is rarely affected since the motherhas not yet developed the antibodies, but all subsequent fetuses are atrisk for disease if the mother is not appropriately treated.

The current treatment for this condition is strictly preventive. Thestrategy is to attempt to keep the woman from initially developinganti-Rh antibodies. This is done by administering 300 μg of animmunoglobulin (Ig) preparation that contains anti-Rh antibodies at 28weeks of gestation and again within 72 hr of birth. This is highlyeffective in preventing the disease when the patient comes in early forprenatal care. Unfortunately, large numbers of women do not obtainproper prenatal care for various reasons and go on to develop stronganti-Rh immune responses. For these women, in utero transfusion of thefetus under ultrasound guidance is the only current treatment availablefor high-risk cases when the woman has previously developed a strongimmune response against the Rh antigen. Because eighty-five percent ofthe Caucasian population is Rh⁺, a considerable number of women andtheir offspring are potentially at risk for contracting the disease.

1.2.3 Attempts To Isolate Active Solid-Phase Rh Antigen Have Failed

Unfortunately, attempts to isolate active Rh antigen have beendisappointing, and no successful attempts at preparing bound forms ofthe antigen have been reported. Indeed, a definitive review (Agre andCartron, 1991) reported that Rh antigenic activity was lost aftermembranes are solubilized or transferred onto immunoblot membranes, andmost biochemical methods therefore actually kill the antigenic activitythat identifies and defines the Rh antigen.

Moore et al. (1982) and Plapp et al. (1979) each reported isolation ofsmall amounts of Rh antigen after affinity chromatography ofdeoxycholate solubilized RBC. Plapp et al. (1979) solubilized the cellsin deoxycholate, added the mixture to an affinity column made ofimmobilized anti-Rh antibodies and eluted the bound fraction. Theresulting eluate was active in inhibition of a reaction between Rh⁺ RBCand antibody. Disappointingly, however, extracts from both Rh⁺ and Rh⁻cells inhibited the reaction, with the authors postulating that Rhantigen was merely “hidden” in Rh⁻ cells.

That conclusion, however, was disproved when modem molecular biologymethods conclusively showed that Rho(D) antigen is not present in Rh⁻cells (Agre and Cartron, 1991), and that the Rh antigen polypeptides hadmolecular weights of between 28 and 32 kDa (Agre and Cartron, 1991).Clearly the 7 kDa polypeptide reported by Plapp and coworkers could notbe the Rh antigen polypeptide.

Moore et al. (1982) surface-labeled RBC with ¹²⁵I, reacted the labeledcells with anti-Rh antibodies, washed the cells and dissolved them indeoxycholate. This was passed over a protein A-Sepharose column andcomplexes were isolated after elution. Although they were successful indetecting Rh antigen in acrylamide gel separations of eluted complexesby autoradiography, the amount of Rh protein isolated by their methodwas too low to provide definitive analysis of Rh protein. In fact, thequantities were so small, that no inhibition assays could be performedto ascertain the activity and integrity of the isolated protein.

A report in 1986 suggested that minor amounts of Rh antigen could beisolated in soluble form (Paradis et al., 1986), but unfortunately, thismethod, too, provided a limited quantity of Rh antigen, and thepreparation was contaminated with cytoskeleton components. Attempts byworkers in the field to repeat the method for isolation oflarge-quantities of active Rh antigen were unsuccessful, as wereattempts to couple the soluble form of the antigen to varioussolid-phase supports and maintain antigenicity of the preparation whenadsorbed to solid-phase matrices such as ELISA plates, nitrocellulose,plastic beads, Sepharose, etc. using standard methodologies.

1.2.4 Unavailability of Solid-Phase Rh Antigen Has Limited Hematology

The unavailability of solid-phase (or bound) Rh antigen compositions,and the lack of ability of using contemporary immunoassay methodologiessuch as ELISA and solid-phase antigen assays have confounded the fieldof hematology for many decades.

Because of these limitations, and because no assays for anti-Rhantibodies exist except for time-consuming, cumbersome, non-quantitativeRBC agglutination assays, the fields of hematology, obstetrics andneonatology are severely lacking in this important regard. Theshortcomings of the present methodologies in the area are many.

First, the results are reported as a titer (ie. the highest dilution ofthe serum in question that gives a standard degree of agglutination). Itis commonly understood in the field that titer results are highlysubjective depending on who reads the result. Variations of ±1 tube areaccepted variations due to this subjectivity. Further, it is commonlyknown that a given serum can be given to two different individuals ortwo different laboratories and the reported titers can be dramaticallydifferent. Even if reporting of titers was absolute, the doublingdilutions used would mean that reported results potentially have almost100% error inherent.

For example, suppose that 5 μg/ml antibody would yield an agglutinationtiter of 1:32. This would mean that the patient would need to have 10μg/ml to yield a titer of 1:64. Thus, an amount of antibody of 9.5 μg/mlwould be reported as a 1:32 titer because only 2-fold dilutions aremade. The higher the antibody concentration, the greater the discrepancybecomes, i e., if the titers were reading 200 versus 400 μg/ml, aconcentration of 390 that is interpreted as 200 is greatly underreported by titering.

1.3 Deficiencies in the Prior Art

The isolation of active Rh antigen polypeptides, and in particular, theRh_(o) D antigen-bearing polypeptide, in large quantity has eludedscientists for more than half a century. That it has not been possibleto isolate, store, and immobilize antigenically- (or serologically-)active blood group antigens, and in particular Rh antigen, represents asignificant limitation in the medical arts. Because of theunavailability of large amounts of antigenically-active blood groupantigen proteins, it has been impossible to develop improved assays andmethods for identifying, isolating and purifying specific antibodieswhich recognize these antigens. Likewise, the unavailability of boundforms of serologically-active blood group antigens has prevented thedevelopment of affinity matrices comprising blood group antigens such asRh antigens, ELISA methodologies specific for these antigens, anddevices for the inline purification and removal of anti-blood groupantibodies from solution. Because of the impossibility of isolatingantigenically-active Rh antigens in quantity using conventional methods,development of such methods and compositions have never been available.Moreover, because no method currently exists for the isolation ofantigenically-active Rh antigens, and in particular D antigen, allcurrent analytical procedures in hematology must rely on theavailability of intact RBCs. Standard blood bank practice relies ondoing agglutination assays using defined RBC and patient serum.

During clinical management of previously alloimmunized patients,critical treatment decisions often depend on a combination of symptomsand laboratory results. Knowledgne of the level of anti-D antibody canbe crucially important in determining the management strategy for suchpatients. Unfortunately, there is a wide variation in the results of thedoubling dilution titers reported by laboratories. A recent surbey oflaboratories was done by the College of American Pathologists todetermine the uniformity of results reported for a single standardanti-D serum (College of American Pathologists, 1996). In the survey,1641 participants were given the same anti-D serum and were asked toreport titers. Titer scores varied widely with results ranging betweentiters of 1:2 and 1:2048. Titers of 1:32 or 1:64 were reported by 59.5%of participants and a titer range of 1:16 to 1:128 was reported by 86%of participants. Thus, 14% of laboratories reported titers below 1:16 orabove 1:128 for the identical sample. Such results dramaticallyillustrate the well-known variability of the doubling dilutionagglutination titer method of anti-Rh antibody measurement currently inuse in the medical community and underline the urgent need fordevelopment of a quantitative assay method for Rh blood group antigens,and D antigen in particular.

Therefore, what is lacking in the prior art is the availability ofantigenically-active blood group antigens, and in particular Rh antigenssuch as the D antigen. Also lacking are methods for the isolation andmaintenance of such antigens in serologically-active forms both solubleand bound. What is needed is the availability of quantitative analysesand methods for the determination of Rh antigens in solution,identification and quantitation of anti-Rh antibodies, and methods anddiagnostic kits for the ready determination of both antigen andantibodies specific for blood group antigens such as Rh antigens, and inparticular D antigen. Such methods and compositions would provide arevolutionary advance in the medical arts, particularly in the areas ofhematology, blood banking, transfusion medicine, obstetrics, andneonatology, and would permit fabrication of devices and apparatususeful for the isolation and purification of anti-Rh antibodies fromsolution. Such apparatus would be particularly useful in treatment ofdisorders such as hemolytic disease of the newborn.

2. SUMMARY OF THE INVENTION

The present invention overcomes these and other deficiencies in theprior art by providing novel methods and compositions comprisingserologically- (antigenically-) active blood group antigens, and inparticular, Rh antigens including the D antigen, which may be adsorbedto a variety of solid supports including ELISA microtiter plates,plastic and glass beads, coverslips, sepharose, agarose, and othersolid-phase antigen-presenting supports. Methods are disclosed for thepreparation, storage, and assay of antigenically-active blood groupantigens such as the Rh antigens in both soluble and bound forms. Theinvention also provides compositions and methods comprising anti-bloodgroup antibodies, and in particular, anti-Rh antibodies, such as anti-Dantibodies, as well as methods for isolation, identification, andquantitation of these antibodies. Other aspects of the invention areapparatus and devices for the isolation of anti-Rh antibodies fromsolution, and in particular, methods and compositions for the isolationand removal of anti-D antibodies from a mammal such as a human. Suchmethods and devices find particular utility in the removal of anti-Rhantibodies from the blood of a pregnant female, and in the treatment andprevention of HDN and other related fetal disorders.

2.1 Methods for Stabilizing Antigenically-Active Forms of Blood GroupAntigens

The inventors have demonstrated that the serologic integrity of Rhantigen extracts can be protected from the detrimental effects of saltbuffers by incorporating amphoteric buffers in the isolation protocol.Suitable amphoteric buffers which may be used to successfully store andmanipulate active Rh antigen include, but are not limited to, thosesolutions which have amphoteric properties. Such buffers are well-knownin the art, and include, among others, WRA, glycine, HEPES, MOPS,Bis-Tris, Alanine, and Acetate. The buffers described by Good and Izawa(1972) are also contemplated to be useful in the practice of theinvention.

In an illustrative embodiment, the inventors have utilized theamphoteric buffer WRA to isolate, store, and manipulate active Rhantigen. The buffers are useful in pH ranges of 1 to 6, and are mostpreferred in the range of from about pH 2 to about pH 7, although higherand lower pH ranges may be contemplated to be useful for certainapplications. Concentrations of from about 0.1% to about 5% for WRA,glycine, HEPES, MOPS, Bis-Tris, and alanine are most preferred, as areconcentrations of from about 0.01 M to about 1.0 M for acetate.

The bound (or solid-phase) antigen is very stable in amphoteric buffersand retains serologic activity for extended periods of time. Using theampholyte/glycine buffer, ELISA assays have now been done successfullywith both the human and the rabbit forms of the Rh antigen. Preferredbuffers include ampholytes as those described in U.S. Pat. No.3,485,736, incorporated herein by reference, although any suchamphoteric buffer is contemplated to be useful in the preparation,storage and adsorption of the antigens disclosed herein.

In a preferred embodiment, the amphoteric composition WRA, a novelbuffer formulated by the inventors, has been shown to be useful in thepractice of the methods disclosed herein. The formulation of WRA bufferis disclosed in Example 1. Alternatively, the ampholyte buffers asdisclosed in U.S. Pat. No. 3,485,736 (incorporated herein by reference)are equally useful in the practice of the present invention.

2.2 Compositions Comprising Solid-Phase Blood Group Antigens

In a preferred embodiment the present invention providesserologically-active blood group antigens immobilized onto a solidsupport or substrate. One such family of preferred antigens is the Rhantigens, and a most preferred Rh antigen is the Rh D antigen. The solidsupport or substrate may be, but is not limited to, matrices, columns,chromatographic media, glass or plastic surfaces, acrylic beads, beadedagarose, Sepharose, coverslips, microscope slides, test tubes, vials,bottles, ELISA supports, and the like. When desired, the antigenicpolypeptides may be adsorbed onto such media either by hydrophobicinteraction, or by active crosslinking of the protein antigen(s) to thesolid support or substrate by cyanogen bromide, oxirane, p-nitrophenylchloroformate activation or by any other suitable means known to thoseof skill in the art.

The solid support may be in the form of an apparatus or device whichcomprises a chamber, one or more inlet ports, one or more outlet ports,and a matrix within the chamber to which the antigenically-active formof the protein or peptide is adsorbed or chemically crosslinked. In anillustrative embodiment, the inventors adsorbed Rh D antigen to t-butylHIC beads (Bio-Rad), passed a solution containing anti-D antibodies overthe column, and removed such antibodies from solution via the binding ofthe anti-D antibody to the D antigen adsorbed to the column. Methods arealso provided for washing such a column, device or apparatus to removecontaminating materials, and then subsequently eluting the boundantibody from the antigen matrix using eluants such as chaotropicreagents.

2.3 Novel Methods for Low-pH Adsorption of Antigens to Solid Matrices

The inventors have devised a methodology incorporating two non-obvioussteps that permit the efficient adsorption of Rh antigens to a solidsupport or substrate. The invention provides novel compositionscomprising such solid-phase active Rh antigens, and provides methods anddevices for solid-phase active Rh antigens, and in particular, theadsorption of Rh antigen to glass and plastic beads and to ELISA plates,microtiter dishes, slides, and other substrata.

The first requirement for preservation of Rh antigenicity when Rhpolypeptides are adsorbed to solid supports is to do all manipulationsinvolving the antigen in salt-free organic buffers particularly organicamphoteric buffers). The second requirement is that the antigenadsorption be performed at low pH (a condition that normally denaturesmost protein antigens). In a surprising finding, the inventors havedemonstrated that adsorption of the antigen under conditions where thepH was 1 to 6 was preferred, with a pH range of 2 to 5 being morepreferred, and a pH of 2.4 to 4.5 being most preferred for adsorption ofthe antigenically-active protein to a solid support.

2.4 Methods for Isolation of Anti-Rh Antibodies

Methods are disclosed and claimed for isolating antibodies specific toblood group antigens from solution using the compositions, devices, andapparatus disclosed herein. In particular, these methods are applicableto the isolation of Rh-specific antibodies from solution. In a preferredembodiment, methods for isolating anti-D antibodies from solution isprovided. Preferably the solution is a biological solution, such asblood, serum, plasma, monoclone culture supernatant, tissue culturesupernatant, bacterial culture supernatant, cell fluid, lymph,cerebrospinal fluid, synovial fluid, or any other biological samplewhere the presence of one or more blood group antibodies are suspected.In preferred embodiments, the solution is a biological solution from amammal, and in particular a human or a rabbit, although the inventorscontemplate that other animals such as bovines, equines, porcines,goats, and the like may also provide a source for the particularsolution to be used in practice of the invention. Preferably the animalis human, and more preferably, the animal is a pregnant female.

By contacting the blood of the mammal with a solid-phase Rh antigencomposition of the invention, anti-Rh antibodies may be removed from thesolution by adsorption to the antigenically-active bound Rh antigen. Ina most preferred embodiment, anti-D antibodies are removed from solutionsuch as human blood, plasma, or serum using a device, composition, orapparatus comprising an antigenically-active form of the D antigen.

To facilitate adsorption of the antibody to the antigen, the solid-phaseantigen composition may be incubated in the presence of a solutioncontaining anti-Rh antibodies with agitation for an appropriate timeperiod to permit adsorption of the antibody to the antigen-matrix.

In an alternate embodiment, the contact can be made in the form of acolumn connected to one or more pumping devices, such as a peristalticpump, for example, to enhance the flow rate of the antibody-containingsolution past the solid-phase support comprising the antigen. Thecontact step may be repeated two, three, four or even more than fourtimes with further depletion of antibodies from the solution at eachstep. In a most preferred embodiment, an in-line affinity matrix columnapparatus is contemplated for the isolation and removal of anti-Rhantibodies from the circulatory system of a human.

2.5 Devices and Apparatus for Isolation of Antibodies from Solution

The invention discloses and claims apparatus and devices which comprisethe novel antigenically-active protein antigens of the presentinvention. These apparatus and devices are provided for the isolation ofantibodies specific for the bound antigens from solution. In particular,devices are provided for the removal of Rh antibodies from thecirculatory system of an animal. Most preferably, the animal is apregnant human female whose circulatory system contains anti-Dantibodies.

The availability of a serologically-active solid-phase antigen alsoprovides a means for specific antibody purification so that dramaticallysmaller doses of anti-Rh antibodies could be used for therapies whichcurrently rely on the whole globulin fraction of pooled high-titeredanti-Rh sera. Polyclonal antibodies are known to be much more active indiagnostic assays than the available monoclones. The devices andapparatus of the present invention represent novel and useful means forremoving specific antibodies from solution, and in particular forlowering the titer of anti-Rh antibodies in the circulatory system of ananimal, and in particular, a woman with a high anti-Rh titer.

In conjunction with the preceding method, the inventors also contemplatethe formulation of apparatus and devices for the in-line removal ofanti-(blood group antigen) antibodies from solution, and particularlyfrom the bloodstream of a pregnant human female. In preferredembodiments, such antibodies are anti-Rh antibodies, with anti-Dantibodies being most preferred. In a general sense the devices comprisea chamber having inlet and outlet ports, and contained within such achamber, a composition comprising an immunologically-active immobilizedblood group antigen. The device is then used to adsorb the anti-(bloodgroup antigen) antibodies from solution passed through the device andover the matrix. Such devices may optionally comprise one or more pumpsto facilitate the passage of solution over the matrix. Single ormultiple inlet and outlet ports may be fitted onto the chamber dependingupon the particular application. Such ports may optionally have fittingssuch as Leur-Lok collars for attachment of tubes, hoses, or syringesfitted with a Leur-Lok connection.

The manufacture of in-line devices for the purification of componentsfrom whole blood, serum, plasma, lymph, synovial fluid, etc. iswell-known in the art. Such devices are useful in applications relatingto plasmapheresis. In this process, plasma is separated from blood cellcomponents and passed through a filtration mechanism.

Absorbed antibody matrices such as Sepharose, cellulose, nylon, glass,acrylic, or other plastic, or inert resins, beads, fibers, etc. are allcontemplated to be within the scope of this application when employedfor the removal of antibodies from solution using the novel Rh antigencompositions disclosed herein.

In a preferred embodiment, the use of t-butyl HIC beads coated with Rhantigen in an inline immunoadsorbant filter under conditions of roomtemperature, pH 7.2 to 7.4 for a period of from about 2 to about 4 hr iscontemplated to be useful for the removal of anti-Rh antibodies from abiological fluid such as plasma.

The inventors contemplate that any such device which comprises anantigen-bound to a matrix could be used in an in-line format with aplasmapheresis machine to remove antibodies from plasma of women withhigh levels of anti-Rh antibodies. The plasma would circulate throughthe device, anti-Rh antibodies would bind to the antigen-matrix, and theplasma eluate would then be reinfused into the patient. This would beparticularly useful in quickly lowering the anti-Rh titer of a pregnantpatient if the fetus is in danger from the presence of such circulatingantibodies. The inventors anticipate that the in-line removal ofantibodies from maternal circulation could be used to deplete anti-Rhantibodies making in utero transfusion unnecessary.

In a general sense, an apparatus of the present invention comprises achamber with an inlet port and an outlet port, and an immobilizedantigenically-active blood group antigen composition contained withinthe chamber. The chamber may be of any shape, although cylindricalchambers are preferred. The antigens which may be bound to the solidsupport include one or more blood group antigens such as a D antigen, ac antigen, a C antigen, an e antigen, an E antigen, an A antigen, a Bantigen, or an F antigen, or any other of the blood group antigensdisclosed herein or known to those of skill in the art.

Optionally, the apparatus can further comprise one or more pumps. Asdescribed herein, the solid support may be of any suitable material towhich one or more antigenically-active blood group antigens may beadsorbed. Preferred matrices include, but are not limited to, glass,plastic, acrylate, methylmethacrylate, Sepharose, agarose, nylon, fiber,or glass wool supports. In a preferred embodiment, the protein orpeptide is immobilized under conditions of low pH, with a pH range ofabout pH 6 to about pH I being preferred and a pH range of from about pH2.4 to about pH 4.5 being most preferred. The proteins or peptides maybe immobilized in the presence of an amphoteric or zwitterionic buffersuch as EDTA, WRA, MOPS, HEPES, glycine, alanine, Bis-Propane orBis-Tris. Typically, the concentration of buffer will be on the order offrom about 0.01% to about 5%, or more preferably, from about 1% to about4%.

One such device contemplated by the inventors to be useful in thepractice of the invention is a column such as the FDA-approved devicefor inline absorption of total IgG antibodies known as a Prosorba®column (IMRE Corp., Seattle, Wash.). This column is approximately3″D×4″H and is filled with a matrix of Silica to which is coupledstaphylococcal Protein A. This column is used to absorb all IgG fromplasma, regardless of specificity. Although it is currently approvedonly for use in patients with Idiopathic Thrombocytopenic Purpura (ITP,an autoimmune disease in which antibodies to platelets or antibodies toforeign antigens that are adhered to platelets destroy the platelets),modification of this column using the novel blood group antigensdisclosed herein would provide a device for the specific removal ofparticular blood group antigens from solution.

The inventors propose a modification of such a column for specificremoval of anti-blood group antibodies, and in particular anti-Rhantibodies using the novel antigens of the present invention. While thecapacity of such a blood group antigen-specific column may differ from anative Prosorbae column, one of skill in the art would be able to modifythe column and formulate a blood group antigen-specific column in asimilar fashion. Such a column could contain a single blood groupantigen, or a combination of two or more blood group antigens. Anexample of this column and the general schematic for its use inisolating antibodies from the circulatory system of an animal isillustrated in FIG. 18 and FIG. 19.

In an illustrative embodiment of this aspect of the invention, theinventors created an apparatus which consisted of a 50 ml columncontaining beads coated with the rabbit homolog of the human Rh Dantigen (Rh_(RABBIT) F). From this device, the inventors isolated 90.2mg of purified anti-F antibody. When the size of the column wasincreased to 60 ml of Rh_(RABBIT) F-coated beads, the inventors isolated145.4 mg of purified anti-F anti-body from a solution passed over thecolumn.

The inventors contemplate that a variety of solid phase supportscomprising adsorbed or covalently crosslinked antigenically-active formsof blood group antigens such as the Rh antigens, and in particular the Dantigen could be fashioned in devices similar to these illustrated inFIG. 19A and FIG. 19B to provide the specific removal of Rhantigen-specific antibodies from solution, and particularly, as aninline means of removing antibodies from the circulatory system of ananimal. One such inline means is illustrated schematically in FIG. 18which depicts a varation of plasmapheresis, a method well-known to thoseof skill in the art for separating plasma from the circulatory system ofan animal. Incorporation of a device or apparatus of the presentinvention into such a method would facilitate an ex vivo isolation andremoval of antibodies directly from the bloodstream of an animal usingone or more serologically-active protein antigens coupled to a matrixcontained within a device such as that illustrated in FIG. 19A and FIG.19B.

2.6 Compositions and Methods for Quantitative Assay of Blood GroupAntigens and Antibodies

The invention provides active Rh antigen for use in ELISAs and relatedquantitative methodologies. The availability of active solid-phase Rhantigen now permits the development of quantitative diagnostic protocolsand kits comprising the compositions disclosed herein. By utilizing theRh antigen to coat ELISA plates, prequantified specifically purifiedantibodies may be used to create a standard curve for quantitation of Rhantigens which may now be prepared in large quantity throughconventional or recombinant methods that are well-known to those ofskill in the art. Patient sera may then be diluted as in conventionalagglutination assays, with the results being quantitated using astandard curve in conventional units (typically μg/ml, e.g.). Suchmethods are well-known in the art, and are routinely performed forantigens such a growth hormones, etc. using ELISA, RIA, or relatedtechniques which are also known to those of skill in the art. In animportant aspect of the invention, the availability of the active Rhantigen compositions disclosed herein, now extends the use of ELISA andRIA methodologies for use in detection and quantitation of anti-Rhantibodies. Prior to the present invention, no such tests had beenavailable to clinicians and hematologists.

One aspect of the invention is a composition comprising an isolated andpurified antigenically-active blood group antigen protein or peptide.Such an antigen is preferably a mammalian antigen such as that derivedfrom a human or rabbit. In preferred embodiments, the antigen is an Rhantigen or a rabbit homolog of a human Rh antigen such as a D antigen, ac antigen, a C antigen, an e antigen, an E antigen, an A antigen, a Bantigen, or an F antigen. In an important aspect of the invention, theprotein or peptide is antigenically-active under conditions of low pHsuch as in the range of from about pH 6 to about pH 1. More preferably,the pH is from about pH 2.4 to about pH 4.5. In sharp contrast to theprior art in which antigenically-active Rh antigens were stable insolution for only short periods of time, the antigen compositions of thepresent invention are stable for significantly longer periods of time,such as e.g., for periods of time from at least 4 hours to as much as192 hours or more.

The compositions of the invention may further comprise an amphoteric orzwitterionic buffer such as EDTA, WRA, MOPS, HEPES, glycine, alanine,Bis-Propane or Bis-Tris, and the like. Typically, the buffer is presentat a concentration of from about 0.01% to about 5%.

The peptide antigen compositions may be soluble, or alternatively theymay be immobilized onto a solid support such as a glass, plastic,acrylate, methylmethacrylate, Sepharose, agarose, nylon, fiber, or glasswool substrate or the like. Immobilized peptide antigen compositions areparticularly contemplated to be useful in the formulation of petridishes, test tubes, vials, microscope slides, ELISA plates, microtiterdishes, culture plates and the like to which it is desirable to adsorbor chemically crosslink the novel peptide antigens. Such immobilizedantigen compositions are particularly preferred for the formulation ofimmunoaffinity columns and similar matrices. Once immobilized, thepeptide antigens may be maintained in solution, or alternatively, may bedried and stored in dry form for extended periods of time. In preferredembodiments, the inventors have shown the antigen compositions to bestable for at least 192 hrs without loss of antigenic activity. Suchcompositions find particular utility in the fomulation ofimmunodetection reagents, diagnostic kits, and blood groupantigen/antibody assays.

2.7 Diagnostic Kits, Immunodetection Reagents, and Assays

The present invention provides methods, compositions and kits forscreening samples suspected of containing Rh antigen polypeptides or Rhantigen-related polypeptides, or cells producing such polypeptides. Saidkit can contain a nucleic acid segment encoding an Rh antigenpolypeptide, or an anti-Rh antibody. The kit can contain reagents fordetecting an interaction between a sample and a nucleic acid or antibodyof the present invention. The provided reagent can be radio-,fluorescently- or enzymatically-labeled. The kit can contain a knownradiolabeled agent capable of binding or interacting with a nucleic acidor antibody of the present invention.

The reagent of the kit can be provided as a liquid solution, attached toa solid support or as a dried powder. Preferably, when the reagent isprovided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent provided is attached to a solidsupport, the solid support can be chromatography media, a test platehaving a plurality of wells, or a microscope slide. When the reagentprovided is a dry powder, the powder can be reconstituted by theaddition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that the Rhantigen peptides of the present invention may be employed to detectantibodies having reactivity therewith, or, alternatively, antibodiesprepared in accordance with the present invention, may be employed todetect Rh antigen or Rh antigen-related epitope-containing peptides. Ingeneral, these methods will include first obtaining a sample suspectedof containing such a protein, peptide or antibody, contacting the samplewith an antibody or peptide in accordance with the present invention, asthe case may be, under conditions effective to allow the formation of animmunocomplex, and then detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either an Rh antigen peptide or an Rhantigen-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of Rh antigen or Rh antigen-relatedproteins or peptides and/or antibodies in a sample. Samples may includecells, cell supernatants, cell suspensions, cell extracts, enzymefractions, protein extracts, or other cell-free compositions suspectedof containing Rh antigen peptides. Generally speaking, kits inaccordance with the present invention will include a suitable Rh antigenpeptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.8 ELISAs and Immunoprecipitation Methods

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating Rh antigenic sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins, cells must besolubilized into detergent micelles. Nonionic salts are preferred, sinceother agents such as bile salts, precipitate at acid pH or in thepresence of divalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

The generic protocol is to coat the wells with a sufficient amount of Rhantigen (e.g., about 50 to about 75 μl or more Rh antigen) for asufficient period of time (e.g., from about 4 to about 16 hr), at asuitable incubation temperature (e.g., from about 4° C. to about 37° C.,with room temperature being most preferred). The plates are then washedand may be blocked at this point by adding from about 50 to about 200 μlof blocking agent and incubating for periods of a few minutes to about 4hr at a temperature of from about 4° C. to about 37° C. The plates maythen be washed again one or more times, and the sample containing theantibody is applied. If a radiolabeled antibody or antigen is used, thenthe sample may be washed and then counted directly using a protocol suchas in a RIA.

Alternatively, in the case of ELISAs, the assay procedure typicallyinvolves the addition of an appropriate substrate at a suitableconcentration for detection either with or without added peroxide (e.g.,when using HAP) and subsequent incubation period (usually from a fewminutes to about 4 hr at a temperature ranging from about 4 to about 37°C.), depending upon the particular protocol used. Reagents are added tostop the enzymatic reaction (i.e., 100 μl of 2 M H₂SO₄). The plates arethen read on an ELISA reader at a wavelength appropriate for theenzyme-substrate system being used. Such protocols are well-known tothose of skill in the art, and may be modified as necessary to includethe novel antigen composition of the present invention.

This same assay can be used to determine serum levels of each of thefour subclasses of IgG. There are some recent papers that suggest thatseverity of symptoms of HDN may be due to differences in thequantitative levels of IgG subclasses (Iyer et al., 1992; Garner et al.,1995). For such an assay, specific conjugates would be used that areeach specific for one of the subclasses. There are murine Mab availablespecific for each of the four human IgG subclasses. These would be usedfor such assays after conjugation to appropriate enzyme.

This same assay is used for creation of a standard curve. For this, apreparation of specifically purified anti-Rh antibody is used that hasbeen precisely quantitated using spectrophotometry or other method.Aliquots of this preparation that have been accurately diluted are putinto the ELISA protocol and a standard curve is generated by plottingantibody concentration in μg/ml versus OD. The particular wavelengthused for optical density measurements will depend upon the particularsubstrate being assayed.

Once the standard curve has been generated for a given batch of Rhantigen, patient samples are read on the linear part of the curve andextrapolated to μg/ml of antibody contained in whole serum.

2.9 Compositions for Western Blots and Related Immunoblot Methods

The antigen compositions of the present invention will find great use inimmunoblot or Western blot analysis. The novel Rh antigens may be useddirectly as standards (e.g., as a positive control) in Western analyseswherein one desires to determine the presence of Rh antigens in a testsample. Alternatively, the novel Rh antigens may be used to isolate,quantitate, purify, and concentrate anti-Rh antibodies which may also beused in Western analyses as a high-affinity primary antibody reagent forthe identification of Rh proteins immobilized onto a solid supportmatrix, such as nitrocellulose, nylon or combinations thereof. Inconjunction with immunoprecipitation, followed by gel electrophoresis,these may be used as a single step reagent for use in detecting antigensagainst which secondary reagents used in the detection of the antigencause an adverse background. This is especially useful when the antigensstudied are immunoglobulins (precluding the use of immunoglobulinsbinding bacterial cell wall components), the antigens studiedcross-react with the detecting agent, or they migrate at the samerelative molecular weight as a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies directed against theanti-blood group antibody are considered to be of particular use in thisregard.

2.10 Compositions for Determining Blood Group Antigen Epitopic CoreSequences

In one aspect, the present invention provides for the first time theability to isolate and purify substantial amounts of Rh protein, and tostore and manipulate this protein without significant loss of antigenicactivity. Prior to the discovery by the inventors that significantlong-term storage and stabilization of the blood group antigens such asthe D antigen could be facilitated by using amphoteric buffers such asWRA, glycine, and the like, it was not possible to isolate or maintaineither in solution or in bound form significant quantities ofantigenically-active blood group antigens. Likewise, prior to thesurprising finding by the inventors that conditions of low pH could beused to bind antigenically-active forms of Rh antigen to solid supports,it was not possible to prepare such compositions. Thus it was notpreviously possible to obtain significant quantities of these antigensto characterize antigenic domains, to determine epitopic core sequences,or to prepare mutated, truncated, or otherwise altered amino acidsequences defining a whole or a portion of an Rh antigen protein.

However, in light of the teaching of the instant specification, it isnow possible to isolate such proteins and to characterize antigenicdomains and epitope sequences therefrom. Thus, the invention disclosesand claims Rh proteins, Rh protein derivatives, and Rh-derived peptidecompositions, free from total cells and other peptides, which comprise awhole or a portion of a purified Rh antigen protein or peptide whichincorporates an epitope that is immunologically cross-reactive with oneor more anti-Rh antibodies.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-Rh antibodies” isintended to refer to a peptide or protein antigen which includes aprimary, secondary or tertiary structure similar to an epitope locatedwithin an Rh antigen polypeptide. The level of similarity will generallybe to such a degree that monoclonal or polyclonal antibodies directedagainst the Rh antigen polypeptide will also bind to, react with, orotherwise recognize, the cross-reactive peptide or protein antigen.Various immunoassay methods may be employed in conjunction with suchantibodies, such as, for example, Western blotting, ELISA, RIA, and thelike, all of which are known to those of skill in the art.

The identification of Rh antigen immunodominant epitopes, and/or theirfunctional equivalents, is a relatively straightforward matter. Forexample, one may employ the methods of Hopp, as taught in U.S. Pat. No.4,554,101, incorporated herein by reference, which teaches theidentification and preparation of epitopes from amino acid sequences onthe basis of hydrophilicity. The methods described in several otherpapers, and software programs based thereon, can also be used toidentify epitopic core sequences (see, e.g., Jameson and Wolf, 1988;Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence ofthese “epitopic core sequences” may then be readily incorporated intopeptides, either through the application of peptide synthesis orrecombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of 8 to 20 amino acids in length, and morepreferably about 8 to about 15 amino acids in length. It is proposedthat shorter antigenic Rh antigen-derived peptides will provideadvantages in certain circumstances, for example, in the preparation ofvaccines or in immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution. Previously, it was not possible to isolate significantamounts of Rh protein, to maintain such protein in solution for extendedperiods of time, to prepare peptide fragments derived from Rh antigen,or to identify or characterize the epitope(s) present on the protein.The availability of Rh antigen protein in quantity provides anopportunity to prepare immunogenic Rh compositions which maintain theirserologic integrity for extended periods of time.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to Rh antigen and Rhantigen-related sequences. These epitopic core sequences are identifiedherein in particular aspects as hydrophilic regions of the Rh antigenpolypeptide antigen. It is proposed that these regions represent thosewhich are most likely to promote T-cell or B-cell stimulation, and,hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind anantigen binding site. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of the Rh peptide in theamphoteric buffers disclosed herein, the Rh peptide compositions may bereadily stored in an amphoteric buffer such as WRA, glycine, Bis-Tris,MOPS, HEPES, Tris, etc. for fairly long periods of time if desired,e.g., up to six months or more, without appreciable degradation or lossof antigenic activity. Where extended aqueous storage is contemplated itwill generally be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4° C., or more preferably, frozen. Of course, where the peptides arestored in a lyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse. The Rh antigen may be stored in a lyophilized state either byitself, or alternatively, may be bound to a solid support prior todrying. The inventors contemplate that the antigen may be stored in adry form either bound to beads, matrices, or prepared onto an ELISAplate or other suitable support depending upon the particularapplication for which it will be used for periods of time extending fromweeks to months.

2.11 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons listed in Table 1.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, ie., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (4.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.12 Methods for Producing Anti-Blood Group Antigen Antibodies

An important aspect of the invention relates to the generation ofantibodies which are reactive against either a whole or portion of an Rhantigen peptide isolated as described herein. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g., Harlowand Lane, 1988; incorporated herein by reference). The methods forgenerating monoclonal antibodies (mAbs) generally begin along the samelines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance with the present invention andcollecting antiserum from that immunized animal. A wide range of animalspecies can be used for the production of antiserum. Typically theanimal used for production of antiserum is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies. Alternatively, antiserum may be obtained from ahuman subject.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of antibody is obtained, the immunizedanimal can be bled and the serum isolated and stored, and/or the animalcan be used to generate mAbs. mAbs may be readily prepared through useof well-known techniques, such as those exemplified in U.S. Pat. No.4,196,265, incorporated herein by reference. Typically, this techniqueinvolves immunizing a suitable animal with a selected immunogencomposition, e.g., a purified or partially purified Rh antigen protein,polypeptide or peptide. The immunizing composition is administered in amanner effective to stimulate antibody producing cells. Rodents such asmice and rats are preferred animals, however, the use of rabbit, sheep,or frog cells is also possible. The use of rats may provide certainadvantages (Goding, 1986, pp. 60-61), but mice are preferred, with theBALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of the animal with the highest antibody titerwill be removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used for fusion, as areknown to those of skill in the art (Goding, pp. 65-66, 1986; Campbell,pp. 75-83, 1984). For example, where the immunized animal is a mouse,one may use P3-X63/Ag8, X63- Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with human cellfusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Arninopterin and methotrexate block de novo synthesis of both purinesand pyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants-(after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted, cloned, andpropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. Hybridoma cells can be injected(often into the peritoneal cavity) into a histocompatible animal of thetype that was used to provide the somatic and myeloma cells for theoriginal fusion. The injected animal develops tumors secreting thespecific monoclonal antibody produced by the fused cell hybrid. The bodyfluids of the animal, such as serum or ascites fluid, can then be tappedto provide mAbs in high concentration. The individual cell lines couldalso be cultured in vitro, where the mAbs are naturally secreted intothe culture medium from which they can be readily obtained in highconcentrations. mAbs produced by either means may be further purified,if desired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography.

2.12 Means for the Removal of Anti-Rh Antibodies from Solution

Another aspect of the present invention concerns methods for removinganti-Rh antibodies from solution. In a general sense, the methodinvolves the immobilization of a serologically-active blood groupantigen, such as an Rh antigen, as disclosed herein, and passing asolution containing antibodies reactive thereto over the immobilizedantigen under conditions which permit the binding of specific antibodiesto the bound antigen. Such a process may comprise a solid-phase matrixonto which one or more antigenically-active blood group antigen proteinshave been fixed. In an illustrative embodiment, the matrix comprisesplastic beads (10 to 100 μm in diameter) such as Bio-Rad MacroPrephydrophobic interaction chromatography (HIC) beads, and in particulart-butyl HIC beads, although the inventors contemplate a variety ofsimilar beads or matrices may be used to immobilize the active antigen.In practice, any suitable matrix to which the antigen may be adsorbed,fixed upon, or crosslinked to is desirable, as long as the serologicintegrity and antigenic property of the Rh antigen is maintained. Themethod for removing antibodies from solution in a general sensecomprises passing a solution suspected of containing Rh antibodies overthe matrix under conditions which allow the formation ofantigen-antibody (immune) complexes. The efficiency of the process maybe monitored by comparing the titer of Rh antibodies in solution beforeand after the solution is passed over the matrix, or alternatively, theantibodies which were bound to the matrix may be eluted from the columnor matrix and collected.

The solution from which antibodies may be removed include, but are notlimited to, physiological fluids, such as blood, lymph, serum, synovialfluid, cerebrospinal fluid, plasma, culture supernatant, tissue culturesupernatant, monoclone culture supernatant, etc. or any other biologicalfluid in which the presence of Rh antibodies is suspected. Preferablythe solution is blood, and more preferably, mammalian blood. In apreferred embodiment, the inventors contemplate the method to be usefulin the in-line removal of anti-Rh antibodies from the bloodstream of ahuman female which is suspected of containing Rh antibodies. In general,the offspring of a female with an anti-Rh titer in excess of 1:2 who ispregnant by an Rh⁺ male are considered to be at risk for HDN if theoffspring are also Rh⁺. The first child from such a pregnancy may be atrisk for HDN if there was significant transplacental bleeding early inpregnancy, and if the mother developed anti-D antibodies as a result ofthe first pregnancy. Subsequent offspring from such a pregnancy are alsoat risk for HDN. This is critical, because such fetuses may suffersevere clinical problems due to the maternal antibodies attacking anddestroying fetal RBCs. In fact, such conditions may lead to severeanemia in the fetus, and in some cases fetal intrauterine death due to adepletion of fetal RBCs.

In a preferred embodiment, a method of purifying an Rh antibody isdisclosed. The method generally comprises contacting a sample suspectedof containing an antibody with an immobilized antigen under conditionseffective to bind the antibody and subsequently eluting the antibodyfrom the immobilized antigen.

A method of removing an Rh antibody from a biological fluid is alsodisclosed and claimed in the invention. The method generally comprisescontacting such a fluid with an immobilized Rh antigen under conditionseffective to bind the antibody to the antigen.

2.13 Definitions

The current literature uses two nomenclatures to express genetic andseologic information on the human red blood cell Rh blood group antigensystem. The Rh-Hr terminology derives from the work of Wiener (1943) whobelieved that the immediate gene product is a single entity called anagglutinogen. According to Wiener's concept, each agglutinogen wascharacterized by numerous individual serologic specificities calledfactors, each of which was recognized by its own specific antibody.

The CDE terminology was introduced by British workers Fisher and Race(1948) and it reflected the concept that individual genes determinedeach antigen. The same letter designation was used for both the gene andthe gene product in the Fisher-Race system, except that, by convention,the symbols for genes were always listed in italics. Both nomenclatureshave remained in use today, although recent molecular biology advanceshave shown that each major antigenic specificity in the Fisher-Racenomenclature system in encoded by a distinct gene, and that five genes,D, C, c, E, and e encode five phylogenetically-related, but distinct,peptide antigens which are termed D, C, c, E, and e.

Thus, as used throughout this specification, a blood group antigen isintended to mean any protein or peptide antigen present on the surfaceof a red blood cell. For example, an Rh blood group antigen is intendedto mean a D antigen, a C antigen, a c antigen, an E antigen, an eantigen, or any other related Rh antigen. A blood group antigen is alsointended to mean a Rh_(o), rh′hr′, hr″ or rh″ antigen according to theearlier nomenclature of Wiener. A blood group protein is intended tomean any protein or peptide present on the surface of a red blood cellthat contains within its sequence one or more antigenic regions to whichanti-blood group antigen antibodies will bind. An Rh protein or peptideis intended to mean a D protein or peptide, a C protein or peptide, a cprotein or peptide, an E protein or peptide, an e protein or peptide, orany other related Rh blood group protein or peptide. A blood groupprotein is also intended to mean an Rh_(o), rh′hr′, hr″ or rh″ proteinor peptide according to the earlier nomenclature of Wiener. A bloodgroup antigen is also intended to mean an amino acid sequence whichdefines a portion or a whole of a gene product derived from a D gene, aC gene, a c gene, an E gene, or an e gene. Moreover, a blood groupprotein is also intended to mean any mammalian-derived antigen which ishomologous to any of these human antigens. Such antigenic proteinsinclude, but are not limited to, the rabbit A, D, and F antigens.

Likewise, an anti-(blood group antigen) antibody is intended to mean anantibody which specifically recognizes and binds to an antigen presenton the surface of a red blood cell. An anti-Rh antibody is intended tomean an antibody such as an anti-D antibody, an anti-C antibody, ananti-c antibody, an anti-E antibody, an anti-e antibody, or any otherrelated Rh antibody. An anti-(blood group antigen) antibody is alsointended to mean an antibody which specifically recognizes and binds toan antigen present on the surface of a red blood cell such as ananti-Rh₀ antibody, an anti-rh′ antibody, an anti-rh″ antibody, ananti-hr′ antibody, or an anti-hr″ antibody following the nomenclature ofWiener. Similarly, an anti-(blood group protein) is also intended tomean any mammalian-derived antibody which is specific for any protein orpeptide antigen which is homologous to a human blood group antigen. Suchantibodies include, but are not limited to, the rabbit anti-A, anti-D,and anti-F antibodies.

In addition to the two major blood group systems (ABO and Rh) thefollowing blood group antigen systems are known, and are also consideredto be within the scope of the present invention: MNSsU, P1, Lutheran,Kell, Lewis, Duffy, Kidd, Diego, Cartwright, Dombrock, Colton, Scianna,Xg^(a), I/i, Augustine, Cromer, En^(a), Gerbich, Gregory, Holley,jacobs, Joseph, Lngereis, Ok^(a), Vel, Chido, Rodgers, Cost-Stirling,York, Knops-Helgeson, McCoy, John Milton Hagen, Ahonen, Batty, Biles,Bishop, Box, Chr^(a), Dantu, Froese, Good, Griffiths, Heibel, Hey, Hov,Hunt, Jensen, Jn^(a), Lewis II, Livesey, Mitchell, Moen, Orriss, Peters,Radin, Redelberger, Reid, Rosennlund, Swann, Torkiidsen, Traversu, Webb,Wright, Wulfsberg, and Bg. Each of these systems may have severalantigens and phenotypes, but all have at least one known antigen (e.g.,Lewis has two antigens and 3 phenotypes).

3. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1. Aliquots of human D⁺ antigen were dialyzed overnight vs. 0.1 MPBS or against 0.5 M NaHCO₃ overnight followed by 4 hr dialysis vs. PBS.The samples were then used to inhibit a reaction between monoclonalanti-D and trypsin-treated D⁺ human RBC in a slide agglutination assay.Untreated Cx is the original antigen preparation in EDTA buffer and theUninhib. Cx is the antibody mixed with RBC with only PBS added in placeof inhibitor.

FIG. 2. Aliquots of Rh_(RABBIT) antigen were put in the presence of theindicated final concentrations of several proteins, ampholytes,detergents, zwitterions, etc. for 16 hr. And the inhibitory capacity ofthe mixture on an RBC slide agglutination reaction was measured. EDTABuffer was used as the Untreated Cx. and the Uninhibited Cx. was madeusing only EDTA buffer as a volume control.

FIG. 3. Aliquots of Rh_(RABBIT) antigen were put in the presence of theindicated final concentrations of amphoteric buffers for 16 hr. and theinhibitory capacity of the mixture on an RBC slide agglutinationreaction was measured. Rh_(RABBIT) antigen in EDTA buffer was used asthe EDTA control (top) and EDTA buffer as a volume control in anuninhibited control (bottom) was used.

FIG. 4. Rh_(RABBIT) antigen in EDTA buffer was made to finalconcentration of 0.2 M glycine by adding dry glycine. The pH of onealiquot was then dropped to 2.4 with 6 M Hcl, a second aliquot wasraised to pH 11 using ethylenediamine, and the pH of a third aliquot wasraised to pH 7.4 also using ethylenediamine. One volume of each aliquotof Bio-Rad HIC t-butyl beads was mixed with two volumes of Rh_(RABBIT)antigen and incubated over-night with agitation. The OD₂₈₀ of thesupernatant and washes was read in a spectrophotometer and compared tothe total OD₂₈₀ of antigen added to beads to determine the amount ofantigen bound to beads.

FIG. 5. Rh_(RABBIT) antigen in EDTA buffer was mixed with aliquots ofbeads as described in FIG. 4 with the pH adjusted to the values shown.After incubation and washing, the OD₂₈₀ of supernatants was determinedas in FIG. 4.

FIG. 6. Results of six studies adsorbing Rh_(RABBIT) antigen to HICbeads at neutral pH in EDTA buffer and 10 separate studies adsorbing in0.2 M Glycine, pH 2.4. Absorption and OD₂₈₀ determinations were done asin FIG. 4.

FIG. 7. HIC beads were incubated in Glycine buffers ranging from pH 1.0to 7.3 over-night with agitation, and washed in EDTA Buffer, pH 7.4until the pH of each set was at 7.4. Rh_(RABBIT) antigen in EDTA Buffer,pH 7.4 was added and the mixtures were agitated overnight. The sets ofbeads were then centrifuged and washed 3 times. Each set of supernatantsfrom each bead set was measured for volume and OD₂₈₀ and the totalpercentage of antigen adsorbed to bead sets was determined bysubtraction.

FIG. 8. Three types of plastic beads and glass beads were coated withrabbit Rh antigen in glycine buffer at pH 2.4 as described in FIG. 4.All bead types were approximately 50 μm diameter. Followingcentrifugation and supernatant analysis the percentage of Rh antigenadsorbed to beads was determined by subtraction.

FIG. 9. Bio-Rad t-butyl HIC beads were coated with Rh_(RABBIT) Fantigen. One aliquot of naked (uncoated) beads and 2 aliquots ofRh_(RABBIT) F-coated beads were prepared. An aliquot of each antisenun(anti-Rh_(RABBIT) A and anti-Rh_(RABBIT) F) was prepared to give anagglutination titer of 1:640 with homologous RBC. Aliquots ofanti-Rh_(RABBIT) F were mixed with either control beads (Cx. beads) orRh_(RABBIT) F-coated beads. An aliquot of anti-Rh_(RABBIT) A was alsomixed with Rh_(RABBIT) F-coated beads. The resulting supernatants wereassayed by slide agglutination assays.

FIG. 10. Antigen extracts from both D⁺ and D⁻ RBCs were prepared. Eachextract was then coated on t-Butyl HIC beads at pH 2.4 in Glycinebuffer. Two monoclonal anti-D antibodies were absorbed with either D⁺antigen-coated beads or D⁻ antigen-coated beads. Supernatants were thenexamined in slide agglutination assays with Ficin treated D⁺ RBCs.

FIG. 11. Bio-Rad t-Butyl HIC beads were coated with rabbit F antigen atpH 2.4, 7.4 and 11.0 and washed. Control beads (No Coat) were also used.Anti-F serum was diluted to give a titer of 1:1,280 after absorptionwith naked beads (bottom bar in FIG. 11). After absorption of identicalaliquots of anti-F with beads coated at pH 2.4, 7.4 and 11.0, thesupernatants were assayed by slide agglutination assays using trypsintreated rabbit F RBC.

FIG. 12. Bio-Rad t-Butyl HIC beads were coated with rabbit F antigen.Aliquots of beads were then mixed with anti-F antiserum, washed, andtreated with three eluting agents (3 M NH₄ thiocyanate, pH 4.5; 200mg/ml chloroquin diphosphate; NaOH solution at pH 11.5). Supernatantswere exhaustively dialyzed against PBS and each supernatant was thentitrated using trypsinized F RBC in a slide agglutination assay.

FIG. 13. Bio-Rad t-Butyl HIC beads were coated with rabbit F antigen. Itwas determined that aliquots of beads had adsorbed a total of 0.85 OD₂₈₀units of Rh antigen (top bar). The supernatant of an aliquot of beadswhich had been mixed with anti-F anti-bodies and eluted with 3 Mthiocyanate had an OD₂₈₀ of 0.526 following dialysis in PBS (second barfrom top) and beads coated only with Rh_(RABBIT) F antigen (no anti-Fadded) and naked bead supernatants had negligible OD₂₈₀ after dialysis(bars 3 and 4 from top).

FIG. 14. Antibody titers of 3 M thiocyanate eluates from aliquots ofBio-Rad t-Butyl HIC beads in FIG. 11 following exhaustive dialysis vs.PBS. Titers were determined using trypsinized Rh_(RABBIT) F RBC in aslide agglutination assay.

FIG. 15. Optical density of eluates from FIG. 11. After dialysis vs. PBSdetermined spectrophotometrically.

FIG. 16. Enzyme linked immunoadsorbent assay using polystyrene platescoated with either Rh⁺ or Rh⁻ antigen extracts in 0.2 M glycine buffer,pH 2.4. Monoclonal human anti-D was used as test antibody.

FIG. 17. Enzyme linked immunoadsorbent assay using polystyrene platescoated with rabbit F antigen in 0.2 M Glycine buffer, pH 2.4. Rabbitallo-anti-F was used as test antibody.

FIG. 18. Schematic diagram showing the apparatus and methods of thepresent invention used for the removal of blood group-specificantibodies from solution. In a preferred embodiment, the methods anddevices are used for the inline removal of Rh antigens from solution. Asolution suspected of containing Rh antibodies (in this case, acirculatory system of an animal) is passed over the column and the Rhantibodies are bound to the column and removed from the circulatingfluid.

FIG. 19A. Side view of an example of the apparatus of the invention forthe removal of antibodies from solution. The apparatus comprises achamber with one or more inlet and one or more outlet ports throughwhich the solution is passed. Within the chamber is a matrix to whichone of the disclosed serologically-active blood group antigens isadsorbed. This schematic illustrates a device which is contemplated bythe inventors for use in the removal of specific blood group antibodiesfrom solution using the serologically-active antigen-bound matrices ofthe present invention. In one embodiment, the device is used to purifyantibodies which bind to the matrix. This may be done by washing thecolumn after binding to elute contaminating substanecs, and then thesubstantially-purified antibodies may be eluted from the device using anappropriate eluant.

FIG. 19B. Cross-sectional view of an example of the apparatus of thepresent invention. Shown is a chamber having an inlet and an outletport, and a matrix within the chamber to which is adsorbed the novelantigenically-active blood group antigens of the invention. Theapparatus may optionally further comprise one or more pumps forsupplying a sample to the matrix for the removal of anti-blood groupantibodies. The matrix may contain one or more blood group antigens asdisclosed.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 Some Advantages of the Invention

Those of ordinary skill having the benefit of this disclosure willappreciate that the invention provides a number of advantages, includingthe following:

4.1.1 Isolation and Quantitation of Antigenically-Active Blood GroupAntigens

The present invention provides new and important methods of isolating,preparing storing, and quantitating antigenically-active blood groupantigens, proteins, and peptides derived therefrom, and in particular,Rh antigens such as the Rh_(o) D antigen. Also provided are novelmethods of identifying, quantitating, and removing anti-blood groupantibodies, and in particular, anti-Rh antibodies from solution. In amost preferred embodiment, anti-Rh_(o) D antibodies are identified,quantitated and removed from solution using the novel antigens describedherein.

4.1.2 Availability of Solid-Phase Blood Group Antigens

The present invention provides the first successful adsorption ofserologically active blood group antigens, and in particular, active Rhantigens such as D antigen to a solid support or substrate, and inparticular plastic beads and ELISA plates. Surprisingly, the inventorshave shown that more of the antigen is adsorbed/bead at low pH than whenthe adsorption is done with the antigen in a neutral extraction buffer.These results were quite surprising and unexpected for the blood groupantigen peptides, since it is well-known in the art that most proteinsare denatured in low pH solutions, and that most peptide antigens areantigenically inactive (serologically-inactive) below neutral pHconditions. That the inventors have demonstrated Rh antigen activity ina pH range of from about 6 to about 1, and most preferably at a pH offrom about 2.4 to about 4.5 represents a breakthrough in the preparationof solid-phase RBC antigens, and solid-phase Rh antigens in particular.

4.1.3 Isolation of anti-Rh Antibodies from Solution

The inventors have also demonstrated that Rh antigen bound underconditions of low pH absorbs more antibody/jig of antigen extractsolution than when the antigen is adsorbed at standard neutral pH.Moreover, chaotropic reagents (e.g., 3 M NH₄SCN) can successfully elutebound antibodies from antigen-coated solid supports or substrates.

The present invention also encompasses methods for using thecompositions disclosed herein in highly efficient techniques for the invitro and ex vivo isolation and purification of anti-Rh IgG antibodies.The methods and compositions of the invention can be used to producehigh yields of Rh-specific antibodies. In preferred embodiments, Rho Dantigen compositions are used to isolate and purify anti-Rh_(o) Dantibodies from solution. Such methods are most useful in the area oftreating HDN, and in the removal of anti-Rh antibodies from thebloodstream of a pregnant female.

4.1.4 Active Rh Antigen Provides Alternatives to Treatment of HDN

Liley (1963) introduced the method of intraperitoneal intrauterinetransfusion for the human fetus affected by severe alloimmune Rhantibodies. In utero therapy has now advanced to the point of usingultrasound guided umbilical vessel puncture with direct i transfusion ofRBC to the anemic fetus (Bang et al., 1982). Despite this therapy, fetalmortality rates of 10-25% continue to be reported (Grannum et al., 1986;Berkowitz et al., 1986).

The availability of devices and methods for the removal of antibodiesfrom the bloodstream of a pregnant female obviates the need for suchtransfusion methods and greatly reduces the likelihood of HDN. Byreducing the titer of Rh antibodies in the bloodstream throughabsorption to active immobilized Rh antigen compositions disclosedherein, the concentration of anti-Rh antibodies may be significantlyreduced in the mother. In a conventional scenario where intrauterinetransfusion is used to reduce the anti-Rh titer, typically 6 or moretransfusions are performed with such procedures costing in excess of$50,000. The inventors contemplate the inline reduction of Rh antibodiesfrom the mother's circulatory system using the devices and compositionsdisclosed herein to be substantially less expensive with an estimatedfetal saving of 10-15%.

4.1.5 Methods and Compositions Provide Rabbit A, D, and F RBC Antigens

It is clear that the rabbit A, D and F blood group antigens arephylo-genetically equivalent to the human Rh blood group antigen system,and therefore, the present invention also provides methods andcompositions for rabbit RBC antigen isolation, purification, as well asidentification and quantitation of rabbit RBC antigens and antibodies insolution. Kellner and Hedal (1953) showed that the antigens wererestricted to the RBC and that there was no detectable secretion of theminto any body fluids, similar to the human Rh antigen. Additionally,Cohen (1982) pointed out that, due to the complexity of these antigensand their ability in the heterozygous state to create complex antigens,that they must be polypeptides rather than simple sugar residues placedin strategic terminal positions by a gene-controlled transferase. Thecompound antigens created by different combinations of A, D, or Fantigens have been studied extensively in the rabbit (Cohen, 1982; Cohenand Tissot, 1974) and compound antigens derived from combinations ofdifferent Rh antigens have been described for the human (Zaleski et al.,1983) as well. Further, the A, D, and F antigens in rabbits can mediateHDN (Anderson, 1956) as does the Rh antigen in humans.

4.2 “Carter Rh Antigen” Preparations Are Actually Devoid of Antigen

In early papers by Carter and coworkers, methods were described forisolating the “Rh hapten” from red blood cells. Unfortunately, the workis now generally considered invalid, as scientists have been unable toidentify or purify Rh antigen using the disclosed methods. In fact, thepresent inventors were unable to isolate Rh antigen using any of themethods in the prior art as taught by Carter and coworkers. Nineseparate attempts were made to repeat the Carter work. The inventorsused the exact method of extraction as described in the earlyliterature: five times with ether and four times with dichloromethane.All extracts obtained had the color and physical properties consistentwith the published reports, but all such preparations were uniformlydevoid of Rh antigen. No activity was seen even when the inventorsperformed the inhibition assays described in the Carter protocols(Carter, 1949). In no case was any inhibition seen. Therefore,previously reported methods proved unreliable in isolating solubleactive Rh antigen.

4.3 Therapeutic and Diagnostic Kits Comprising Rh Antigen Compositions

Therapeutic kits comprising, in suitable container means, an Rh antigencomposition of the present invention in a pharmaceutically acceptableformulation represent another aspect of the invention. The Rhcomposition may be native Rh antigen, truncated Rh antigen,site-specifically mutated Rh antigen, or Rh antigen-encoding peptideepitopes, or alternatively antibodies which bind native Rh antigen,truncated Rh antigen, site-specifically mutated Rh antigen, or Rhantigen -encoded peptide epitopes. In other embodiments, the Rh antigencomposition may be nucleic acid segments encoding native Rh antigenpolypeptides, truncated Rh antigen polypeptides, site-specificallymutated Rh antigen polypeptides, or Rh antigen-encoding peptideepitopes. Such nucleic acid segments may be DNA or RNA, and may beeither native, recombinant, or mutagenized nucleic acid segments.

The kits may comprise a single container means that contains the Rhantigen composition. The container means may, if desired, contain apharmaceutically acceptable sterile excipient, having associated withit, the Rh antigen composition and, optionally, a detectable label orimaging agent. The single container means may contain a dry, orlyophilized, mixture of an Rh antigen composition, which may or may notrequire pre-wetting before use.

Alternatively, the kits of the invention may comprise distinct containermeans for each component. In such cases, one container would contain theRh composition, either as a sterile polypeptide antigen or polypeptideantibody solution or in a lyophilized form, and the other containerwould include the solid matrix, which may or may not itself bepre-wetted with a sterile solution, or be in a gelatinous, liquid orother syringeable form.

The kits may also comprise a second or third container means forcontaining a sterile, pharmaceutically acceptable buffer, diluent orsolvent. The presence of any type of pharmaceutically acceptable bufferor solvent is not a requirement for the kits of the invention. The kitsmay also comprise a second or third container means for containing apharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, testtube, flask, bottle, syringe or other container means, into which thecomponents of the kit may placed. The matrix may also be aliquotted intosmaller containers, should this be desired. The kits of the presentinvention may also include a means for containing the individualcontainers in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desiredvials, columns, microtiter plates, particulate fiber, or glass woolmatrices, affinity columns, inline adsorption devices, syringes, tubes,or other containers are retained.

Irrespective of the number of containers, the kits of the invention mayalso comprise, or be packaged with, a device for in-line or ex vivoplacement of the antigen-matrix composition for use in treatment of ananimal. Such an instrument may be a syringe, pipette, column, filtrationresin or similar matrix, or any such medically approved in-linesolid-phase antigen containing antibody purification device.

4.4 Affinity Chromatography

Affinity chromatography is generally based on the recognition of aprotein by a substance such as a ligand or an antibody. The columnmaterial may be synthesized by covalently coupling a binding molecule,such as an antigenic protein, for example an Rh protein, to an insolublematrix such as Teflon™, styrene, glass, plastic, cellulose, polyester,polystyrene, etc. The column material is then allowed to absorb thedesired substance from solution. Next, the conditions are changed tothose under which binding does not occur and the substrate is eluted.The requirements for successful affinity chromatography are:

1) that the matrix must specifically-absorb the molecules of interest;

2) that other contaminants remain unabsorbed;

3) that the ligand must be coupled without altering its bindingactivity;

4) that the ligand must bind sufficiently tight to the matrix; and

5) that it must be possible to elute the molecules of interest withoutdestroying them, or eluting the ligand.

A preferred embodiment of the present invention is an affinitychromatography method for purification of antibodies from solutionwherein the matrix contains adsorbed Rh antigen compositions or peptideepitopes derived from one or more Rh antigen polypeptides, adsorbed to asolid-phase matrix such as Sepharose, Teflon™, nylon, plastic, glass, orother suitable matrix. Suitable types of Sepharose matrices includeCL6B, CL4B, and related resins which are known to those of skill in theart. This antigen-matrix binds the antibodies of the present inventiondirectly and allows their separation by elution with an appropriatereagent such as a chaotropic reagent (e.g., NH₄SCN, NaSCN, KSCN, etc.)Another preferred embodiment of the present invention is an affinitychromatography method for the purification of Rh antigen polypeptidesand peptide epitopes from solution. The matrix may include covalentlycoupled anti-Rh antibodies specifically reactive for the Rh antigenpolypeptides. Means for the coupling of antibodies to solid-phasematrices are well-known to those of skill in the art. Theantibody-matrix facilitates the isolation and separation of Rh antigenpolypeptides from solution by elution with a suitable buffer asdescribed above.

4.5 Immunoassays

As noted, it is proposed that native and synthetically-derived peptidesand peptide epitopes of the invention will find utility as immunogens,e.g., in connection with vaccine development, or as antigens inimmunoassays for the detection of reactive antibodies. Turning first toimmunoassays, in their most simple and direct sense, preferredimmunoassays of the invention include the various types of enzyme linkedimmunosorbent assays (ELISAs), as are known to those of skill in theart. However, it will be readily appreciated that the utility of Rhantigen-derived proteins and peptides is not limited to such assays, andthat other useful embodiments include RIAs and other non-enzyme linkedantibody binding assays and procedures.

In preferred ELISA assays, proteins or peptides incorporating Rhantigens, rRh antigens (recombinant Rh antigens), or Rh antigen-derivedamino acid sequences are immobilized onto a selected surface, preferablya surface exhibiting a protein affinity, such as the wells of apolystyrene microtiter plate. After washing to remove incompletelyadsorbed material, one would then generally desire to bind or coat anonspecific protein that is known to be antigenically neutral withregard to the test antiserum, such as bovine serum albumin (BSA) orcasein, onto the well. This allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antiserum onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantiserum or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antiserum with diluents suchas WRA, glycine, (or other amphoteric buffer), BSA, bovine gammaglobulin (BGG), or short-term exposure to phosphate buffered saline(PBS)/Tween™. These added agents also tend to assist in the reduction ofnonspecific background. The layered antiserum is then allowed toincubate for, e.g., from 2 to 4 hours, at temperatures preferably on theorder of about 4° to about 37° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with aamphoteric or zwitterionic buffer solution such as WRA, glycine, oralternatively, for short-term exposure to the buffer, PBS, PBS-Tween®,or borate solutions are acceptable.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and theamount of immunocomplex formation may be determined by subjecting thecomplex to a second antibody having specificity for the first. Ofcourse, in that the test sample will typically be of human origin, thesecond antibody will preferably be an antibody having specificity forhuman antibodies. To provide a detecting means, the second antibody willpreferably have an associated detectable label, such as an enzyme label,that will generate a signal, such as color development upon incubatingwith an appropriate chromogenic substrate. Thus, for example, one willdesire to contact and incubate the antisera-bound surface with a ureaseor peroxidase-conjugated anti-human IgG for a period of time and underconditions that favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in an amphoteric buffer suchas WRA, glycine, or the like, or alternatively, for a short time in astandard buffer such as PBS or PBS-Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

4.6 Immunoprecipitation

The anti-Rh antibodies of the present invention are particularly usefulfor the isolation of Rh antigens by immunoprecipitation.Immunoprecipitation involves the separation of the target antigencomponent from a complex mixture, and is used to discriminate or isolateminute amounts of protein. For the isolation of cell-surface localizedproteins such as Rh antigen polypeptides, peptides may be solubilizedfrom the RBC membrane using methods that are well-known to those ofskill in the art. General methods of peptide isolation involvedissolving cells in one of several detergents, such as Triton X-100™ oralternatively, dissolving membrane skeletons in SDS, and subsequentlypurifying the Rh polypeptides from solution either by hydroxylapatitechromatography and/or preparative electrophoresis (Agre et al., 1987).

4.7 Western Blots

The compositions of the present invention will find great use inimmunoblot or Western blot analysis. The anti-Rh antibodies may be usedas high-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background. Thisis especially useful when the antigens studied are immunoglobulins(precluding the use of immunoglobulins binding bacterial cell wallcomponents), the antigens studied cross-react with the detecting agent,or they migrate at the same relative molecular weight as across-reacting signal. Immunologically-based detection methods inconjunction with Western blotting (including enzymatically-,radiolabel-, or fluorescently-tagged secondary antibodies against thetoxin moiety) are considered to be of particular use in this regard.

4.8 Pharmaceutical Compositions

In methods involving the administration of antigens or antibodies to ananimal, it is necessary to provide pharmaceutically-acceptablepreparations of such compositions. Likewise, the use of in-line medicaldevices for isolation of anti-Rh antibodies also requires the use ofappropriate medically-acceptable formulations of the compositionsdisclosed herein. In this respect, several classes of pharmaceuticalpreparations may be contemplated. For example, the compositions may beorally administered with an inert diluent or with an assimilable ediblecarrier, or they may be enclosed in hard or soft shell gelatin capsule,or they may be compressed into tablets, or they may be incorporateddirectly with the food of the diet. The compositions may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is such that a suitable dosagewill be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The active compounds may alternatively, be administered intravenously,parenterally or intraperitoneally. Solutions of the active compounds asfree base or pharmacologically-acceptable salts can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically-active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

4.9. Epitopic Core Sequences

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic blood group antigenproteins or peptides which include modified and/or extendedepitopic/immunogenic core sequences which result in a “universal”epitopic peptide directed to Rh antigen and Rh antigen-relatedsequences, or other domains which bind anti-Rh antibodies. It isproposed that these regions represent those which are most likely topromote T-cell or B-cell stimulation in an animal, and, hence, elicitspecific antibody production in such an animal.

To confirm that a protein or peptide is immunologically cross-reactivewith, or a biological functional equivalent of, one or more epitopes ofthe disclosed peptides is also a straightforward matter. This can bereadily determined using specific assays, e.g. of a single proposedepitopic sequence, or using more general screens, e.g., of a pool ofrandomly generated synthetic peptides or protein fragments. Thescreening assays may be employed to identify either equivalent antigensor cross-reactive antibodies. In any event, the principle is the same,i.e., based upon competition for binding sites between antibodies andantigens.

Suitable competition assays that may be employed include protocols basedupon immunohistochemical assays, ELISAs, RIAs; Western or dot blottingand the like. In any of the competitive assays, one of the bindingcomponents, generally the known element, such as the Rh antigen -derivedpeptide, or a known antibody, will be labeled with a detectable labeland the test components, that generally remain unlabeled, will be testedfor their ability to reduce the amount of label that is bound to thecorresponding reactive antibody or antigen.

As an exemplary embodiment, to conduct a competition study between an Rhantigen and any test antigen, one would first label an Rh antigen with adetectable label, such as, e.g., biotin or an enzymatic, radioactive orfluorogenic label, to enable subsequent identification. One would thenincubate the labeled antigen with the other, test, antigen to beexamined at various ratios (e.g., 1:1, 1:10 and 1:100) and, aftermixing, one would then add the mixture to an antibody of the presentinvention. Preferably, the known antibody would be immobilized, e.g., byattaching to an ELISA plate. The ability of the mixture to bind to theantibody would be determined by detecting the presence of thespecifically bound label. This value would then be compared to a controlvalue in which no potentially competing (test) antigen was included inthe incubation.

The assay may be any one of a range of immunological assays based uponhybridization, and the reactive antigens would be detected by means ofdetecting their label, e.g., using streptavidin in the case ofbiotinylated antigens or by using a chromogenic substrate in connectionwith an enzymatic label or by simply detecting a radioactive orfluorescent label. An antigen that binds to the same antibody as an Rhantigen polypeptide, for example, will be able to effectively competefor binding to and thus will significantly reduce Rh antigen binding, asevidenced by a reduction in the amount of label detected.

The reactivity of the labeled antigen, e.g., an Rh antigen composition,in the absence of any test antigen would be the control high value. Thecontrol low value would be obtained by incubating the labeled antigenwith an excess of unlabeled Rh antigen, when competition would occur andreduce binding. A significant reduction in labeled antigen reactivity inthe presence of a test antigen is indicative of a test antigen that is“cross-reactive”, i.e., that has binding affinity for the same antibody.“A significant reduction”, in terms of the present application, may bedefined as a reproducible (ie., consistently observed) reduction inbinding.

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated e.g., using recombinatorial chemical technology, to mimic thekey portions of the peptide structure. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and hence are also functional equivalents. Theseinclude compositions which mimic the stable active Rh⁺ antigen complex.The generation of a structural functional equivalent may be achieved bythe techniques of modeling and chemical design known to those of skillin the art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

5. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1

Isolation of Active Rh Antigen

5.1.1 Isolation of RBC Soluble Fraction

All operations were carried out at 4° C. unless noted otherwise. LysisBuffer consisted of 0.01 M Tris-HCl, pH 7.4. Triton Buffer was made byadding Triton X-100 to a final concentration of 10% (wt/wt) in LysisBuffer. EDTA buffer consisted of 0.1 mM EDTA (pH 8.0). Optionally, theprotease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added at afinal concentration of 10 μg/ml, although the inventors saw nodifferences in results using buffer from which PMSF was omitted.

RBC were washed at least 5 times in saline. The packed RBC pellet wassuspended in 20 volumes of precooled Lysis Buffer and incubated on icefor 20 min with occasional agitation. The lysate was then centrifuged at20,000 rpm (48,000×g) in a Sorvall centrifuge using an SS-34 (8×50 ml)rotor for 15 min at 4° C. Both centrifuge and rotor were precooled to 4°C. The resulting membrane pellets were washed 4 times with 10 volumes ofLysis Buffer. The final pellet was then resuspended to the originalpacked RBC volume in Lysis Buffer.

One volume of the membrane suspension was made to a final 0.5%concentration of Triton-X-100® using Triton buffer and the equationgiven below. The solution was incubated for 15 min on ice withoccasional agitation, then centrifuged for 10 min at 20,000 rpm asabove. The pellet was washed at least four times with 10 volumes ofLysis Buffer to remove the residual detergent, and the last supernatantwas then discarded.

The pellet was resuspended in 2 volumes of prewarmed EDTA Buffer, andincubated for 30 min at 37° C. with occasional agitation, and thencentrifuged at 20,000 rpm (48,000×g) for 15 min. The pellet was thendiscarded and the supernatant was retained for further use. The initialvolume of packed RBC above was defined as 1 volume. Thus, 10 ml ofpacked RBC were lysed in a total of 200 ml Lysis Buffer and the washesusing 10 volumes would be 100 ml, etc.

0.1X=0.005(Y+X)  Equation 1

where X is the volume of the 10% Triton Buffer solution to add and Y isthe volume (in ml) of the membrane suspension used.

5.2 Example 2

Determination of Activity of Rh Antigen

5.2.1 Enzyme Treatment of Rabbit RBC

Rabbit blood was collected in anticoagulant (Heparin, 20 U/ml), andcentrifuged at 2,000 rpm (900×g) for 15 min at 4° C. The RBC pellet wasresuspended and washed at least 3 times in normal saline (0.85% NaCl).In a tube containing 2.4 ml of PBS and 280 μl of a 1:3 dilution of BactoTrypsin (Difco) (equivalent to 200 μg/ml trypsin) and 100 μl packed,washed RBC were added. This mixture was incubated for 10-15 min at 37°C., then centrifuged and washed at least 3 times in normal saline. Thepellet was then resuspended in a total volume of 5 ml to make a 2%suspension.

5.2.2 Ficin Treatment of Human RBC

Human Rh⁺ blood was collected from a blood bank unit in anticoagulant(citrate-phosphate-dextrose) then centrifuged and washed in PBS. One mlof packed RBC was mixed with 1 ml of PBS in a test tube. In a secondtube, 2.25 ml PBS and 37.5 mg of Ficin (Sigma Chemical Co., St. Louis,Mo.) was mixed gently. The tubes were incubated separately for 15 min at37° C. then the contents of the two tubes were mixed and incubated foran additional 15 min. Cells were then centrifuged and washed three timeswith PBS, and resuspended to a concentration of 2% for use inagglutination assays. Trypsin digestion was utilized in rabbit RBCs andFicin in human RBCs to expose more of the epitopes which are targets forantibodies, thereby making the assays more sensitive.

5.2.3 General Antigen Extract Analysis Protocol

An inhibition assay was performed for antigen analysis usingenzyme-treated RBCs in a 2% suspension in phosphate buffered saline(PBS). The standard antiserum was diluted 1:10 in PBS with 2% normalserum (NS), then 2-fold dilutions were made (i.e., 1:10, 1:20, 1:40,1:80, etc.). Equal volumes of each diluted serum aliquot and antigenextract adjusted to 1.3 OD₂₈₀ (1 cm path length) were mixed andincubated for 1 hr at 37° C. Controls consisting of EDTA buffer withoutantigen extracts were prepared in similar fashion. A 50 μl aliquot ofeach mixture was placed on a glass slide and 50 μl of a 2% RBCsuspension in PBS was added. Slides were rotated slowly for 8 min on aLabLine 3D rotator, the highest dilution recording a weak agglutination(anything that appears more agglutinated than the negative control) wasscored as the titer.

If the antigen preparation contains active Rh antigen, the titer on theset of tubes mixed with the antigen extract is lower than the titer inthe control set due to the binding of soluble antigen and antibodyduring the 1 hr incubation, and the concomitant reduction in antibodyavailable for agglutinating the enzyme-treated RBCs (FIG. 3).

5.2.4 Antigen Destruction by Metal Salt-Based Buffers

Initial studies showed that the antigenic activity of the Rh antigen wasrapidly destroyed when the antigen was exposed to salt buffers. In FIG.1 the inhibition capacity of the antigen was shown to be destroyed after16 hr in PBS or in 0.5 M sodium bicarbonate/PBS (a standard buffer forELISA coating). Additional studies on the stability of the isolatedantigen are described below in section 5.9.

5.2.5 Rh Antigen Activity is Buffer Dependent

In FIG. 2, Rh antigen was incubated in the presence of the indicatedfinal concentrations of several buffers, proteins, ampholytes,detergents, zwitterions, etc. for 16 hr and the inhibitory capacity ofthe mixture on an RBC agglutination reaction was measured. If the addedcomponent had no effect on the antigenicity of the Rh antigen, noagglutination would be expected. If the buffer destroyed or diminishedRh antigen antigenicity, an increase in the level of agglutination wouldbe expected. Using EDTA buffer as the 0% (positive) control and areaction lacking any Rh antigen as the 100% (negative) control, theleast destruction of Rh antigen was observed when either 0.5% finalconcentration of polyethylene glycol 6000 or 2% WRA buffer (pH 7.4) wasused to buffer the Rh antigen preparation.

WRA (wide-range ampholyte) buffer was prepared frompentaethylene-hexamine (PEHA) (Aldrich Chemical Co., #29,275-3) andacrylic acid (99+%, Aldrich Chemical Co., #14,723-0) which had beenrendered devoid of hydroxyquinone monomethyl ether (HMME). HMME wasremoved from the commercial source of acrylic acid by passage through abed of Amberlyst A-27 (Rohm and Haas, Philadelphia, Pa.; Aldrich#21,643-7) according to the manufacturer's instructions at a ratio of 10ml Amberlyst per 100 ml of acrylic acid.

139.2 ml PEHA was then slowly added to 210 ml of previously-degassed (byboiling) distilled water with constant stirring. 130.34 ml of purifiedHMME-free acrylic acid was then added to the flask, which was thensealed and gassed thoroughly with dry medical-grade nitrogen and stirredfor 16 hr at a constant temperature of 70° C. The solution was thenallowed to cool to room temperature. Distilled water was added to bringthe total volume to 673 ml, making a 40% (wt/vol) solution. Thissolution was stored at 4° C. in a brown bottle. In this synthesis, 3.02moles of acrylic acid was used per mole of PEHA, or 1 mole acrylic acidper 2 moles of PEHA amino groups. Thus approximately 50% of the PEHAamino groups in WRA buffer were substituted with an acid group.

In typical studies, the 40% stock solution of WRA buffer was diluted toa concentration of 3% using distilled water giving a pH of approximately6.6-7.0. The final working pH was modified using either ethylene diamine(to raise the pH) or propionic acid (to lower the pH).

Since WRA is an amphoteric organic buffer, several other amphotericbuffers were analyzed. FIG. 3 shows comparative results of using severalbuffers, all at concentrations of 3% compared to the EDTA buffer whichserved as the 100% uninhibited control. Clearly, all the amphotericbuffers tested including WRA, glycine, HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) and MOPS(3-[N-morpholino]propanesulfonic acid), all at pH 7.4 equally preservedRh antigen activity.

5.3 Example 3

Methods for Preparing Immobilized Rh Antigen

5.3.1 Coating Beads

Glass or plastic beads (50 μm diameter) were washed in EDTA buffer, pH2.4 and the supernatant was discarded. One ml of bead pellet was mixedwith 2 ml Rh antigen of 1.3 OD₂₈₀ units (1 cm path length) which wasmade 0.2 M in glycine by adding dry glycine and the pH was lowered to2.4 using 6 M HCl, and the mixture is agitated for 16-48 hr at 4° C. Themixture was centrifuged and washed 3 times, the total volume and finalOD₂₈₀ of each supernatant was collected and measured. The total proteinrecovered in the washes was determined and compared with the totalamount of Rh antigen added for determination of adsorption percentage.

5.3.2 Coating ELISA Wells

ELISA wells are coated with 50-75 μl of Rh antigen which was made 0.2 Mwith respect to glycine and the pH was lowered to 2.4 using 6 M HCl.Plates were incubated for various times (4-16 hr) at varioustemperatures (room temperature or 4° C.) and the wells were washed withappropriate buffers in preparation for the next reagent.

5.3.3 Results

Studies on pH dependence for coating plastic beads was analyzed. FIG. 4shows that strict pH dependence was shown with low pH being thepreferred pH for coating Rh antigen onto the surface of plastic beads(t-Butyl HIC beads were used). Additional studies with unit pHincrements between pH 5.0 and 1.0 confirmed that the peak adsorptionefficiency was reached around pH 2.0 (FIG. 5). FIG. 6 shows that theranges of adsorption efficiencies for 6 separate preparations of Rhantigen isolated in EDTA buffer at pH 7.4 do not even overlap with therange of adsorption efficiencies for 10 different lots of Rh antigenprepared in glycine buffer at pH 2.4. These data confirmed theimportance of low pH conditions for the successful preservation ofactive Rh antigen when immobilizing the antigen to a solid support.

5.3.4 pH Effects are on Rh Antigen and Not on the Adsorbing Matrix

Since many matrices commonly used in the immobilization of antigens areplastic, it was important to determine whether or not the use of suchlow pH solutions for the immobilization of the active antigen had aneffect on the composition of the matrix itself. Polymethylmethacrylateis a polyester, and since esters are known to be hydrolyzable at extremepH ranges, it was critical to demonstrate that the low pH dependence ofRh antigen adsorption was due to pH effects on the Rh antigen itself,and not merely chemical alteration of the matrix, causing it to becomechemically reactive or otherwise modified.

Studies were performed to demonstrate that the low pH used during theimmobilization step did not alter the plastics used as matrices. Nakedsets of beads were treated with glycine buffers ranging from pH 1.0 to7.3 overnight with agitation, and then washed in EDTA Buffer (pH 7.4)until the pH of each set was 7.4. Rh antigen in EDTA Buffer, (pH 7.4)was then added and the mixtures were agitated overnight. The sets ofbeads were then centrifuged and washed 3 times. Each set of supernatantsfrom each bead set was measured for volume and OD and the totalpercentage of antigen adsorbed to bead sets was determined bysubtraction. The results of the study are shown in FIG. 7. Regardless ofpretreatment pH between pH 7.3 and 1.9, all bead sets adsorbed about thesame percentage of Rh antigen. Thus, the heightened adsorption capacityof Rh antigen on plastic beads at pH 2.4 was an effect on the proteinitself, and no an effect of chemically altering the matrix to bind moretotal protein.

5.3.5 Matrix Compositions Useful for Rh Antigen Adsorption

To demonstrate the wide-range of preferred matrices for use in thepractice of the present invention, three different types of plasticbeads and glass beads were coated with Rh antigen in glycine buffer atpH 2.4 as described above. All bead types were approximately 50 μmdiameter. Following centrifugation and supernatant analysis thepercentage of Rh antigen adsorbed to beads was determined bysubtraction. FIG. 8 shows that glass beads adsorbed 56% of Rh antigen,the Bio-Rad SM-2 (polystyrene divinylbenzene, catalog #152-3924)adsorbed 62%, Bio-Rad MPE (polymethyl methacrylate) adsorbed 80% and theBio-Rad 1-Butyl HIC (t-butyl polymethyl methacrylate, catalog #156-0090)were the most efficient, adsorbing 95% of the Rh antigen added.

5.4 Example 4

Methods for Determining Activity of Bound Antigen

The activity of Rh antigen bound to the surface of a solid matrix wasdetermined qualitatively by absorption of anti-Rh antibody from serum. A2-ml aliquot of serum was diluted to a known titer (i.e., 1:512). One mlof this was mixed with 1 ml packed beads coated with the Rh antigenpreparation and 1 ml was mixed with 1 ml packed naked control beads. Themixture was agitated for 2 hr and centrifuged. The supernatant of eachsample was then used in a direct agglutination assay. The decrease intiter of the aliquot incubated in the presence of Rh antigen-coatedbeads was compared to the control.

5.5 Example 5

Methods for Determining Specificity of Bound Rh Antigen

5.5.1 Adsorbed Blood Group Antigens are Immunologically Specific

It is crucial that any antigen coated on a solid surface retain itsactive conformation, and hence its antigenic specificity if it is to beused for immunoassay or antibody purification purposes. To demonstratethat the novel adsorption methods described herein for immobilizingactive Rh antigen did not alter its specificity, studies were conductedwhich demonstrated the effectiveness of the disclosed methods inpreserving antigen specificity. To demonstrate that the methods werealso not limited to just one particular blood group antigen, or to onespecies of animal, two different rabbit antigens and the human Rh Dantigen were assayed for specificity following antigen immobilizationonto solid support matrices such as plastic beads.

5.5.1.1 Rabbit Allelic RBC Antigens A and F

In one study, plastic beads were coated with rabbit F antigen. Onealiquot of naked (uncoated) beads and 2 aliquots of F antigen-coatedbeads were prepared. An aliquot of each antiserum (anti-A and anti-F)was prepared to give an agglutination titer of 1:640 with homologousRBCs. Aliquots of anti-F were mixed with either control beads (Cx.beads) or F antigen-coated beads. An aliquot of anti-A was also mixedwith beads coated with F antigen. The results are shown in FIG. 9.

F antigen-coated beads absorbed anti-F antibodies but not anti-Aantibodies (FIG. 9, top 2 bars). Control beads (uncoated) did not absorbanti-F antibodies (FIG. 9, bar 3). Control agglutination reactions areshown in the bottom two bars. Thus, naked beads do not absorb anti-Fantibodies, and A antigen-coated beads do not absorb anti-F. Only Fantigen-coated beads absorb anti-F antibodies. Thus, antigen specificityis retained after immobilization to a solid substrate.

5.5.1.2 Human Rho(D) Antigen

Antigen extracts from both Rho(D)⁺ and Rho(D)⁻ RBCs were prepared.Results demonstrated that the D⁺ extract inhibited a reaction of each oftwo monoclonal anti-D reagents with D⁺ ficin-coated RBC but the D⁻extract did not inhibit the reactions. Each extract was then coated ont-Butyl HIC beads at pH 2.4 in glycine buffer and washed. Each mAb hadan agglutination titer of 1:1,280 with D⁺ RBCs (FIG. 10, bars 3 and 6from the top). When aliquots of mAb1 and mAb2 were mixed with aliquotsof D⁺ extract-coated beads, the titers of supernatants dropped to 1:40(FIG. 10, bars 1 and 4 from the top). When identical aliquots of mAb1and mAb2 were mixed with identical aliquots of D⁻ extract-coated beadsthe titers were unchanged from control levels (FIG. 10, bars 2 and 5from the top). Thus, antigen specificity was also maintained afteradsorption of human blood group antigens to a matrix.

The conclusion from both the rabbit and human specificity studies isthat the antigens do not become non-specifically sticky for randomantibodies upon adhering to a solid phase support. Instead,immunological specificity is retained.

5.5.2 Adsorbed Blood Group Antigens Retain a High Degree of Antigenicity

Although it was shown in 5.5.1 that antigen extracts from an RBCpopulation (as defined by OD₂₈₀) remain active and specific whenadsorbed to a solid support in a pH-dependent manner, the inventors havealso demonstrated that the adsorbed protein retains a high degree ofantigenic activity even after undergoing a low-pH adsorption.

Bio-Rad t-Butyl HIC beads were coated with rabbit F antigen at pH 2.4,7.4 and 11.0 and washed. Naked beads were used as a control. Anti-Fserum was diluted to give a titer of 1:1,280 after absorption with nakedbeads (FIG. 11, bottom bar). After absorption of identical aliquots ofanti-F with beads coated at pH 2.4, 7.4 and 11.0, the supernatants gaveagglutination titers as shown in FIG. 11. Although beads coated at pH11.0 and 7.4 removed some antibodies, the beads coated with an antigencomposition at pH 2.4 absorbed the anti-F antibodies completely. Thisshows that the antigen composition coated at pH 2.4 is clearly moreantigenically-active than the antigen composition coated at pH 7.4 or11.0 in absorbing specific antibodies from antiserum.

5.6 Example 6

Isolation of Blood Group Antigen-Specific Antibodies

5.6.1 Methods for Isolating anti-Rh Antibodies Using Immobilized RhAntigen

To demonstrate the utility of the disclosed antigen compositions inremoving specific antibodies from solution, devices and methods weredeveloped which permit the ready isolation of antibodies specific forparticular blood group antigens from solution. In a preferredembodiment, such devices and methods are useful for the removal ofanti-Rh antibodies from the plasma of a pregnant female.

In these studies, one volume of t-Butyl HIC beads was coated with 2volumes of Rh antigen and washed in EDTA buffer, pH 7.4. The coatedbeads were then packed into a column to provide a device for the removalof antibodies from solution. One volume of an immune serum solution waspassed through the column and then the column was washed with 0.1 Mglycine, pH 7.4, until the OD₂₈₀ dropped to a baseline level. Severalvolumes of 3 M ammonium thiocyanate were passed through the device andthe eluate was recovered, dialyzed exhaustively against PBS to removethe thiocyanate, and then quantitated by spectrophotometry at 280 nm todetermine protein content.

In the routine practice of the invention, the inventors havedemonstrated that the serum used may either be intact serum,concentrated serum, or diluted in any of the zwitterionic buffersdisclosed herein. The protocol may also be used for either polyclonal ormonoclonal antibodies.

5.6.2 Compositions for Eluting Antibodies from Devices

In addition to the methods described in 5.6.1, the inventors havedemonstrated that various eluting buffers may be used in the routinepractice of the invention to recover antibodies once bound to thesolid-phase antigen compositions.

In sharp contrast to the teachings of the prior art, however, theinventors have found that low-pH glycine buffer (a universally-usedmeans of eluting antibodies from solid state supports) was completelyineffective as an elution agent using the compositions, methods, anddevices described herein. Elution attempts with low-pH glycine bufferseluted almost no detectable antibodies.

To that end, the inventors investigated the use of chaotropic reagentsand high-pH solutions for successful elution of the antibodies. In oneparticular study, Bio-Rad t-Butyl HIC beads were coated with rabbit Fantigen and aliquotted into three test tubes. Anti-F antiserum was addedto each aliquot of beads, and the beads were thoroughly washed bycentrifugation. Each aliquot of beads was then treated with threeeluting agents (3 M ammonium thiocyanate, pH 4.5; 200 mg/ml chloroquindiphosphate; NaOH solution at pH 11.5). Supernatants were exhaustivelydialyzed against PBS and each supernatant was then titrated usingtrypsinized F-containing RBCs. These data (shown in FIG. 12) showed thatnaked beads mixed with antibody did not absorb any antibody elutablewith thiocyanate, and that thiocyanate was the most efficient eluant.

In this study, HIC beads were coated with antigen in EDTA buffer, pH7.4, and it was determined that 56% of the antigen bound to the beads.One ml coated beads were mixed with 2 ml anti-F having a neat titer of1:2,560 and the beads were then centrifuged. Antibodies were eluted with4 ml of 0.2 M glycine, pH 2.4 at 37° C. for 2 hr. The eluate wasexhaustively dialyzed versus PBS. Agglutination assays of this firsteluate were weakly positive only with undiluted eluate. Supernatanttiter of serum after mixing with beads was 1:160 compared with a control(absorbed using 1 ml naked beads) that was 1:1,280. The same beads weremixed with a second aliquot of fresh anti-F after washing them in WRAbuffer. These beads were mixed with 1 ml of anti-F diluted to a titer of1:256 in WRA buffer for 1 hr at 37° C. These beads were centrifuged andwashed in WRA buffer. The beads were incubated at 37° C. for 2 hr in 4ml of 0.2 M glycine, pH 2.4. After centrifugation, the supernatant wasexhaustively dialyzed against PBS. Agglutination assays of the secondsupernatant after dialysis were negative. Supernatant titer of serumafter mixing with beads the second time was now 1:80 indicating that asmall additional amount of antibody was absorbed the second time butthat beads were likely saturated with antibody since some of theantibody did not bind. Due to dilution, the second supernatant wouldhave been 1:128 if no antibody had been absorbed.

The clear result was that 0.2 M glycine, pH 2.4 eluted virtually noantibody from antigen-coated, beads which were shown to bind antibody.This was in sharp contrast to the data obtained when chaotropic and/orhigh pH buffers were utilized. This method represents the first knownincidence of an antigen-antibody system which appears to benon-susceptible to the acid elution methods routinely used in the artfor the elution of antibodies from a solid antigen support system.

5.7 Example 7

Methods for Determining Stability of Immobilized Antigen

5.7.1 Stability of Bound Rh Antigen in the Presence of 3 M Thiocyanate

It was important to determine if the adsorbed blood group antigenremained adhered to the solid support during antibody elution steps,since elution of both Ab and antigen would create undesirableantigen-antibody complexes in solution, and would not result inseparation of the antibody from its corresponding antigen.

Bio-Rad t-Butyl HIC beads were coated with rabbit F antigen. It wasdetermined that aliquots of beads had adsorbed a total of 0.85 OD₂₈₀units of antigen (FIG. 13, top bar). The supernatant of an aliquot ofbeads which had been mixed with anti-F antibodies and eluted withthiocyanate had an OD₂₈₀ of 0.526 following dialysis in PBS (FIG. 13,second bar from top), while supernatants from either antigen-coatedbeads lacking antibody treatment or naked control beads alone hadnegligible OD₂₈₀ after dialysis (FIG. 13, bars 3 and 4 from top). Theseresults indicated that chaotropic buffers (such as 3 M ammoniumthiocyanate) successfully eluted antibodies from antigen-coated beads,but did not co-elute the antigen itself.

5.7.2 Antibody Yield After Thiocyanate Elution, Titer and Mass

From these data it was clear that protein (defined as OD₂₈₀) was elutedfrom antigen-coated beads after mixing those beads with antiserum andthat no protein was eluted from antigen-coated beads which had not beenincubated previously with antiserum. The supernatants from each of thethree eluates were tested by agglutination versus trypsinized F RBCsafter dialysis against PBS. FIG. 14 shows that the thiocyanate 5, eluatefrom beads coated at pH 2.4 had a titer of 1:32 whereas the pH 7.4eluate had a titer of 1:2 and the pH 11 eluate had no detectableantibody content. Eluates of naked beads also had no detectableagglutination titer. Thus, eluate from beads coated with antigencompositions at pH 2.4 retained substantial antibody activity.

The protein content of each sample was determined by measuring theoptical density at 280 nm (OD₂₈₀). As shown in FIG. 15, the supernatantof the pH 2.4 aliquot had an OD₂₈₀ of 1.948 and the other supernatantshad dramatically lower OD₂₈₀. This confirms that the eluate recovered inthe pH 2.4/thiocyanate elution protocol had both the highest antibodyactivity and the highest total mass recovered of the three samples.

5.8 Example 8

ELISA Methods and Compositions

5.8.1 ELISA Compositions for Blood Group Antigens

In these studies, all adsorption of blood group antigens onto solidsurfaces was done in 0.1 M glycine buffer, pH 2.4. All washing stepswere done preferably using 0.1 M glycine buffer, pH 7.4, although saltbuffers such as PBS were equally useful for shortened protocols wherebound antigen was not in contact with the buffer for periods longer than4-6 hr. Wash steps optionally included the addition of 0.05-1% Tween-20to the buffer. A blocking step is also optional, but if used, theblocking agent may be any of a whole range of commonly-used agents suchas bovine serum albumin, human serum albumin, hen egg ovalbumin, andmilk proteins such as casein, etc. The developing anti-Ig antibody maybe of goat, sheep, or rabbit, etc. origin. It should be broadly specificfor IgG Fc of all 4 subclasses but for some applications might bespecific for IgG₁, IgG₂, IgG₃, or IgG₄. The antibody may be conjugatedwith horseradish peroxidase, alkaline phosphatase, or any other suitableenzyme. For RIA analysis, the developing antibody may be conjugated with¹²⁵I, ¹³¹I or any other suitable radionuclide.

Standard ELISA plates (Immulon II™) were coated with 50 μl of eitherhuman D⁺ or D⁻ RBC extracts overnight in glycine buffer at pH 2.4.Plates were washed, blocked with 1% human serum albumin, washed, andserial dilutions of mAb1 were added to duplicate wells. After 2 hrincubation, wells were washed and 50 μl of a 1:2,000 dilution ofperoxidase-conjugated goat anti-human IgG (H-chain specific) was added.After 2 hr incubation, the wells were washed and substrate ABTS(Boehringer-Mannheim, 2,2′-azino-di-[3-ethylbenzthiazoline sulfonate]diammonium salt) was added. After 30 min, 50 μl of 2 M H₂SO₄ was addedto stop the reaction. The results are shown in FIG. 16. The D⁻-coatedwells showed only background level reactivity. Thus, human D⁺ antigenextracts effectively coated matrices for use in ELISA methods.

Similar methods were used to coat ELISA plates with rabbit F antigen inglycine, pH 2.4. Peroxidase-conjugated goat anti-rabbit IgG Fc was used.The results shown in FIG. 17 indicated that a typical ELISA formatdilution curve was also obtained when using rabbit blood group antigenextracts.

5.8.2 Analysis of Rh Antigens and Blood Group Antigens M and S

Studies of an antigen composition isolated from human RBCs having thephenotype D, C, c, e, K, k, Fya, Fyb, Jka, Jkb, s, M, N, Pi, Leb wereperformed. The method used was to first coat the wells of polystyreneU-bottom plates with 75 μl of antigen extract in glycine buffer, pH 2.4.Identical plates were incubated 4 hr at 37°, 25° and 4° C. Afterwashing, 50 μl of monoclonal antibody was added at 37° C. for 15 min andthe plates were then washed repeatedly with PBS. 50 μl of ficin-treatedRBCs were then added to each well and the RBCs were allowed to settleout at room temperature. The presence of bound antibody (positivereaction) was determined by adherence of the ficin-treated indicatorcells over the surface of the well due to bridging. The absence ofantibody binding (negative reaction) was indicated by pelleting of theficin-treated RBCs into a pellet in the bottom of the microtiter platewell. The results of the assays are shown in Table 2. The results showthat adsorption efficiency was not temperature-dependent for the rangeof about 4 to about 37° C.

TABLE 2 Analysis of Human RBC Extract and Effects of AdsorptionTemperature 4° C. 25° C. 37° C. Anti-D + + + Anti-C 0 0 0 Anti-c w+ w+w+ Anti-E + + + Anti-e + + + Anti-M 0 0 0 Anti-S 0 0 0 Phenotype: D, C,c, e, K, k, Fya, Fyb, Jka, Jkb, s, M, N, Pl, Leb

Moreover, these data also demonstrated that the D, c, and e antigenswere detected in the antigen extract preparation. While the C antigen(reportedly present on the RBCs tested) was not detected during thisstudy, it is not known whether such inability to detect the C antigenwas due to a pH lability of the antigen itself, or either mis-typing ofthe particular lot of RBCs used in the study. Likewise, the reason forthe unexpected reaction of anti-E may also be due to an initialmis-typing of the RBC preparation, or non-specificity of the anti-Eantibody employed. Regardless, the data show that the present methodsand compositions are successful for a wide-range of Rh blood groupantigens from both animal and human sources, and are not limited to Rh Dantigen alone.

5.8.3 Stability of Immobilized Antigen—Drying and Rehydration Studies

The antigen compositions described herein were also used to coat platesfor drying and rehydration studies. Plates were coated for 4 hr withantigen in glycine, pH 2.4 at 25° C. Plates were washed with PBS andthen 100 μl of Immucor(D drying agent (Immucor, Inc., Norcross, Ga.), 5%WRA anphoteric buffer, or nothing (not even water) was added. The plateswere then patted dry on a paper towel. The plates were placed in a foilpouch (forms a complete water barrier) with desiccant to remove waterand a moisture indicator, sealed, and placed at 4° C. After 48 hr,plates were analyzed using monoclonal antibodies as described above. Theresults shown in Table 3 indicate that the D, c and e antigens wereantigenically equal to freshly prepared plates (column marked “fresh”).

TABLE 3 Effects of Drying on Human RBC Extracts Immucor ® 5% WRA NothingFresh Anti-D + + + + Anti-C 0 0 0 0 Anti-c + + + w+ Anti-E + + + +Anti-e + + + + Anti-Jka 0 0 0 0 Anti-Jkb 0 0 0 0

The only exception was that the c antigen seemed slightly stronger inthe 3 dried plates than in the freshly prepared plate. Again the Eantigen was detected and C was not, as was discussed in the previoussection. These results show that the methods for isolating active bloodgroup antigens give rise to antigenic compositions which may besuccessfully dried and rehydrated 48 hr after coating on plasticsurfaces. Surprisingly, the Kidd blood group antigens present in theoriginal RBC preparation were not detected in the extracts, even whenfreshly-prepared plates were utilized.

Other studies were done to determine the length of time that the driedantigen was stable and could be successfully rehydrated. The plates werecoated with acidified antigen compositions at different concentrations.Wells were coated with concentrations of antigen extracts ranging from500 μg/ml down to 15.6 μg/ml. 100 μl was added/well, and plates weredried as above. The plates were rehydrated and then analyzed asdescribed above.

Results after dry storage for 2 days and for 5 weeks are described inTables 4-9 for each of three preparations of different beginningphenotypes.

These results show that the antigen compositions isolated using themethods described herein from three different phenotypes maintainsantigenic stability for up to 5 weeks after drying. In nearly all casesonly a 1 tube drop in concentration was evident after 5 weeks of totaldryness. The c, e cell antigen compositions showed the greatestspecificity with the least non-specific reactivity. As in other assays,the anti-E reacted nonspecifically with D, C, c, e and D, D, e cellextracts, but not with c, e antigen compositions. This could indicate asimilarity in epitopes of D and E revealed by drying. An alternative isthat the acidic treatment is denaturing E antigen to be nonspecificallyreactive with this particular monoclonal anti-D. Regardless, theclearest result is that antigen compositions can be successfully driedonto the surface of plastic plates and retain major antigenic reactivityafter at least 5 weeks in the dry state. These results indicate asignificant improvement over the current state of the art where stablepre-coated ELISA matrices are not available for blood group antigenssuch as the Rh_(o)(D) factor.

TABLE 4 D, C, c, e Cell Antigen Extract After 48 hr Concentration of AXAdded to Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.5 31.2515.62 Anti-D + + + + +/− 0 Anti-C 0 0 0 0 0 0 Anti-c + + + + + +/−Anti-E + + + + + +/− Anti-e + + + + +/− 0

TABLE 5 D, C, c, e Cell Antigen Extract After 5 Weeks Concentration ofAX Added to Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.531.25 15.62 Anti-D + + + +/− 0 0 Anti-C 0 0 0 0 0 0 Anti-c + + + + +/− 0Anti-E + + + + +/− 0 Anti-e + + + + +/− 0

TABLE 5 D, C, c, e Cell Antigen Extract After 5 Weeks Concentration ofAX Added to Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.531.25 15.62 Anti-D + + + +/− 0 0 Anti-C 0 0 0 0 0 0 Anti-c + + + + +/− 0Anti-E + + + + +/− 0 Anti-e + + + + +/− 0

TABLE 8 c, e Cell Antigen Extract After 48 hr Concentration of AX Addedto Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.5 31.25 15.62Anti-D + +/− 0 0 0 0 Anti-C +/− +/− 0 0 0 0 Anti-c + + + + + + Anti-E+/− 0 0 0 0 0 Anti-e + + + + + +/−

TABLE 8 c, e Cell Antigen Extract After 48 hr Concentration of AX Addedto Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.5 31.25 15.62Anti-D + +/− 0 0 0 0 Anti-C +/− +/− 0 0 0 0 Anti-c + + + + + + Anti-E+/− 0 0 0 0 0 Anti-e + + + + + +/−

TABLE 9 c, e Cell Antigen Extract After 5 Weeks Concentration of AXAdded to Wells During Coating at pH 2.4 (μg/ml) 500 250 125 62.5 31.2515.62 Anti-D 0 0 0 0 0 0 Anti-C +/− 0 0 0 0 0 Anti-c + + + + 0 0 Anti-E0 0 0 0 0 0 Anti-e + + + + + +/−

5.9 Example 9

Methods for Long-Term Storage of Active Rh Antigen

5.9.1 Stability of Antigen Compositions in EDTA Buffer at 4° C.

Several lots of rabbit RBC antigen compositions have been stored in EDTAbuffer for various periods of time at 4° C. and analyzed for theirability to inhibit an Ab-RBC reaction. The results are shown in Table10. These data clearly show that the compositions disclosed are stableat 4° C. for at least 47 days when stored in EDTA buffer.

TABLE 10 Stability of Rh antigen in EDTA Buffer at 4° C. Antigen DateType Prepared Initial Inhibition Later Inhibition Age of Antigen A10/12/95 640 to 160 2,560 to 320 47 days A 10/26/95 640 to 640 2,560 to320 33 days F 11/02/95 640 to 160 2,560 to 160 27 days

5.9.2 Stability of Soluble Rabbit RBC Antigen Compositions

One ml aliquots of rabbit blood group F antigen extract (AgX) was mixedwith an equal volume of the buffers shown in the legend of Table 11.Buffers were initially at a concentration twice that shown in thelegend. In Tables 12 and 13, the pH was adjusted to either pH 4.5 or pH2.4 as indicated. Incubations of the mixtures were done at roomtemperature for the times indicated (4 hr, 24 hr 48 hr, 96 hr, and 192hr). At the end of the incubations, the samples were used as inhibitorsin a standard agglutination assay. In Tables 12 and 13, the pH of theEDTA controls were 4.5 and 2.4, respectively.

TABLE 11 Stability of Rabbit RBC Antigen Extract -- SolubleExtract--Neutral pH Time of Incubation Buffer* 4 hr 24 hr 48 hr 96 hr192 hr WRA 80# 80 80 80 80 Glycine 80 160 160 160 160 HEPES 160 160 320320 640 MOPS 320 320 640 640 640 Bis-Tris 80 80 80 80 160 Bis-Propane160 160 320 320 640 Alanine 160 160 160 160 320 Pharmalyte 80 160 160160 160 Saline 80 160 160 160 320 Borate 1280 1280 1280 1280 1280Phosphate 160 320 320 320 320 Sod. Bicarbonate 1280 1280 1280 1280 1280Acetate 80 160 160 160 320 EDTA/AgX 80 80 80 80 80 EDTA no X 640 640 640640 1280 *Buffers/pH/final concentrations: WRA, wide range ampholytes,pH 7.4, 1.5%, Glycine, glycine-HCl, pH 6.3, 1.5%; HEPES,N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), pH 5.6, 1.5%;MOPS, 3-(N-morpholino)propanesulfonic acid, pH 5.4, 1.5%; Bis-Tris,(bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane), pH 8.8, 1.5%;Bis-Propane, (1,3-bis[tris(hydroxymethyl)methylamino]-propane), pH 9.3,1.5%; # Alanine, alanine-HCl, pH 7.1, 1.5%; Pharmalyte ™, Pharmaciabrand ampholytes, pH 7.1, 1.5%; Saline, NaCl, pH 6.3, 0.85%; Borate,boric acid/NaOH/NaCl, pH 8.0, 0.15 M; Phosphate, NaH₂PO₄/K₂HPO₄, pH 7.0,0.15 M; Sod. Bicarbonate, sodium bicarbonate, pH 8.1, 0.25 M; Acetate,sodium acetate, pH 7.7, 0.15 M. EDTA/AgX is antigen extract in EDTA.EDTA no X is the EDTA buffer only control, no antigen extract added.#Values are inverse titer values, i.e. a 1:80 dilution is entered as 80.

TABLE 12 Stability Of Rabbit RBC Antigen Extract -- Soluble Extract--pH4.5 Time of Incubation Buffer* 4 hr 24 hr 48 hr 96 hr 192 hr WRA 160#320 320 320 320 Glycine 640 1280 1280 1280 1280 HBPES 160 320 640 640640 Bis-Tris 320 640 640 1280 1280 Saline 320 640 640 640 640 Borate1280 1280 1280 1280 2560 Sod. Bicarb. 1280 1280 1280 1280 2560 Acetate<5 <5 <5 <5 <5 EDTA/AgX 80 160 160 160 160 EDTA no X 1280 2560 2560 25601280 *Buffers at the following concentrations, all adjusted to pH 4.5:WRA, Glycine, HEPES, and Bis-Tris at 1.5%, Saline is 0.85% NaCl,Bicarbonate is 0.25 M, Acetate is 0.15 M. #Inverse titer data reportedas described in Table 11. EDTA/AgX is antigen extract in EDTA. EDTA no Xis the BDTA buffer only control, no antigen extract added.

TABLE 13 Stability of Rabbit RBC Antigen Extract -- Soluble Extract--pH2.4 Time of Incubation Buffer* 4 hr 24 hr 48 hr 96 hr 192 hr WRA <5# <5<5 <5 <5 Glycine Hemo- Hemo- Hemo- Hemo- Hemo- lyzed lyzed lyzed lyzedlyzed HEPES <5 <5 <5 <5 <5 Bis-Tris 1280 1280 1280 1280 1280 Saline 25601280 2560 2560 2560 Borate 2560 2560 2560 2560 2560 Bicarbonate 25602560 2560 2560 2560 Acetate <5 <5 <5 <5 <5 EDTA/AgX 640 320 320 640 640EDTA no X 1280 640 640 640 640 *Buffers were at the followingconcentrations, all adjusted to pH 2.4: WRA, Glycine, HEPES, andBis-Tris @ 1.5%, Saline is 0.85% NaCl, Bicarbonate is 0.25 M, Acetate is0.15 M. #Inverse titer data reported as described in Table 11 EDTA/AgXis antigen extract in EDTA. EDTA no X is the EDTA buffer only control,no antigen extract added.

5.9.3 Bound Antigen Studies

Bio-Rad HIC beads, 50 μm, were coated with antigen extract (AgX)overnight in glycine buffer, pH 2.4. After washing in EDTA buffer, a 3ml aliquot of beads was suspended in the Test Buffers and incubated atroom temperature for the indicated times. At 15 the end of incubation,0.5 ml aliquots of packed beads were withdrawn at different times andwashed in EDTA buffer, pH 7.0, and the aliquots were then mixed with anequal volume of antiserum. The tubes containing beads and antiserum wereagitated to thoroughly mix the contents and the tubes were incubated at37° C. for 2 hr. The tubes were then centrifuged and the supernatantswere titered. Data are shown in Tables 14, 15 and 16.

TABLE 14 Stability of Rabbit RBC Antigen Extract Antigen Bound toBio-Rad HIC Beads--Neutral pH Time of Incubation Buffer* 4 hr 24 hr 48hr 96 hr 192 hr WRA 80# 80 80 80 80 Glycine 40 40 40 40 40 HEPES 20 2020 40 40 MOPS 40 40 40 80 80 Bis-Tris 80 80 80 80 80 Bis-Propane 160 320320 320 320 Alanine 40 40 20 20 40 Pharmalyte 160 160 160 160 160 Saline40 80 80 80 80 Borate 160 320 320 320 320 Phosphate 160 160 160 160 160Sod. Bicarb. 640 320 320 320 640 Acetate 20 20 20 20 20 EDTA Std. 20 2040 40 40 Noncoated 640 640 640 320 640 #Inverse titer data reported asdescribed in Table 11. #Values are inverse titer values, i.e. a 1:80dilution is entered as 80. The EDTA Std. is the bound antigen in EDTAbuffer only at neutral pH. Non-coated is the control consisting ofnon-antigen-coated beads, i.e. the negative control.

TABLE 15 Stability of Rabbit RBC Antigen Extract Antigen Bound ToBio-Rad HIC Beads--pH 4.5 Time of Incubation Buffer* 4 hr 24 hr 48 hr 96hr 196 hr WRA 20# 40 10 10 5 Glycine <5 <5 10 10 5 HEPES <5 5 <5 <5 <5Bis-Tris 10 10 5 <5 5 Saline <5 10 <5 5 10 Borate <5 20 10 10 5 Bicarb.5 20 5 5 5 Acetate 20 80 80 80 40 EDTA Std. <5 10 <5 5 10 Non-coated2560 2560 2560 2560 2560 *Buffers at the following concentrations, alladjusted to pH 4.5: WRA, Glycine, HEPES, and Bis-Tris at 1.5%, Saline is0.85% NaCl, Bicarbonate is 0.25 M, Acetate is 0.15 M. The EDTA Std. isthe bound antigen in EDTA buffer at pH 4.5. Noncoated is the control ofnon-antigen-coated beads, i.e. the negative control. #Inverse titer datareported as described in Table 11.

TABLE 16 Stability of Rabbit RBC Antigen Extract Antigen Bound toBio-Rad HIC Beads--pH 2.4 Time of Incubation Buffer* 4 hr 24 hr 48 hr 96hr 192 hr WRA 80# 80 40 20 20 Glycine 2560 1280 2560 320 160 HEPES 80 2020 10 20 Bis-Tris 40 10 5 5 5 Saline 10 10 10 10 10 Borate 10 10 10 1010 Bicarb. 10 20 10 5 5 Acetate 1280 320 640 320 160 EDTA Std. 10 20 1010 20 Noncoated 2560 2560 2560 2560 2560 *Buffers at the followingconcentrations, all adjusted to pH 2.4: WRA, Glycine, HEPES, andBis-Tris @ 1.5%, Saline is 0.85% NaCl, Bicarbonate is 0.25 M, Acetate is0.15 M. The EDTA Std. is the bound antigen in EDTA buffer at pH 2.4.Noncoated is the control of non-antigen-coated beads, i.e. the negativecontrol. #Inverse titer data reported as described in Table 11.

5.9.4 Coating Step Studies

One ml aliquots of Bio-Rad HIC beads, 50 μm, were washed in EDTA buffer.Mixtures consisting of 2 ml of AgX from rabbit F RBC in EDTA buffer and2 ml Test Buffer at 2× concentration at pH 2.4 were made and added towashed beads. The mixture was incubated overnight at 4° C. with constantagitation. The following morning, the tubes were centrifuged. Thesupernatants were saved. The beads were washed 2× with EDTA buffer andthe supernatants were also saved. The beads were then washed 2× with 0.1M glycine buffer, pH 7.0. The bead pellet was then mixed with 1 ml ofanti-F antiserum and incubated at 37° C. for one hour with occasionalagitation. The tubes were then centrifuged and the supernatant wasremoved and titered. Data are shown in Table 17. Thus, a reduction intiter would indicate the presence of functional antigen on the beads andno reduction in titer would indicate the presence of inactive antigen orthat antigen was not absorbed to beads. However, as is readily apparentin Table 8, all preparations adsorbed antigen in three separateexperiments with adsorption amounts ranging from approximately 55-94%.Therefore, the inability of beads coated with antigen using buffersother than WRA or Glycine was due to the antigen coating beingnonfunctional, inactive antigen caused by the chemical nature of thebuffer used.

TABLE 17 Buffer Composition Effects for Coating Bio-Rad HIC Beads at pH2.4 Buffer* Titer# WRA 64 Glycine 32 HEPES 256 Bis-Tris 512 Saline 512Borate 512 Bicarbonate 256 Acetate 512 Noncoated Beads 512 *Buffers atthe following final concentrations, all adjusted to pH 2.4: WRA, GlycineHEPES, and Bis-Tris at 1.5%, Saline is 0.85% NaCl, Bicarbonate is 0.25M, Acetate is 0.15 M. #Inverse titer data reported as described in Table11.

5.9.4 Stability of Soluble Rabbit RBC Antigen Compositions

In general, the soluble antigens were more sensitive to buffervariations than the bound antigen, regardless of pH. At the higher pHranges in Table 11 the antigens are much less stable in salt buffers(borate, phosphate, bicarbonate) than in the other buffers tested. Themost preferred buffers for maintaining the antigenic activity of thesoluble antigen extract at neutral pH were WRA, Bis-Tris, and the EDTAbuffer, although the Pharmacia Pharmalyte buffers were also useful inmaintaining antigenic activity, albeit at a lesser degree than the mostpreferred buffers. Other buffers such as alanine, glycine, HEPES, MOPSand Bis-Propane were more destructive than WRA at neutral pH, but weregenerally less destructive than salt buffers and saline. Thus, WRA,Bis-Tris, and EDTA are the most preferred buffers for maintainingantigenic activity of the soluble form of the antigen extract at theupper end of the pH range.

However, at low pH, significant differences were noted. Only HEPES, WRA,and acetate buffers at pH 2.4, maintain the stability of the antigen(Table 13). All the other buffers, salt based or zwitterionic, damagethe antigen. The results at pH 4.5 are intermediate in most cases,except for acetate which is highly protective even at pH 4.5 (Table 12).

5.9.5 Stability of Rabbit Rh Antigen Compositions During Coating Step

Notably, only WRA and glycine buffers at pH 2.4 provide acceptablechemical conditions for the coating step that results in functionalantigen coated on the beads (Table 17). All other buffers tested destroythe antigen although they allow the antigen to be coated onto the beads(Table 18).

TABLE 18 Quantitative Adsorption of Rabbit F Antigen to Bio-Rad HICBeads at pH 2.4 Buffer* Study 1 Study 2 Study 3 WRA 79.9# 77.2 74.3Glycine 92.1 89.4 94.2 HEPES 90.3 87.2 93.7 Bis-Tris 76.8 54.5 81.2Saline 65.8 72.8 70.8 Borate 72.5 73.0 71.7 Bicarbonate 68.8 75.1 80.4Acetate 82.2 74.4 86.6 *Buffers at the following concentrations, alladjusted to pH 2.4: WRA, Glycine, HEPES, and Bis-Tris at 1.5%, Saline is0.85% NaCl, Bicarbonate is 0.25 M, Acetate is 0.15 M. #Numbers arepercentages of total antigen protein added to the beads which adsorbedto the bead preparation. This was determined spectrophotometrically at280 nm by subtraction of recovered protein from the amount initiallyadded. Three separate studies were performed over the course of threeweeks' time to assess the adsorption

5.9.6 Stability of Rabbit RBC Bound Antigen Compositions

For the most part, the Rh antigen was stabilized after binding to solidsurfaces as compared to the soluble form and is more stable at any pHtested when bound than is the soluble counterpart at a given pH. Withthe notable exceptions of glycine and acetate buffers, the antigenicproperties of the bound Rh antigen compositions are largely unaffectedby the buffers studied (Table 14, 15 and 16). The destructive effects ofglycine and acetate at pH 2.4 were noted to decrease with time.

These data suggest that the Rh antigen compositions of the presentinvention should be stored before use at neutrality in WRA, Bis-Tris ormerely in the EDTA buffer used in the extraction step, be coated ontoplates or beads only in WRA or glycine buffers at pH 2.4, and the boundantigen can then be exposed to biological or immunological reagents atneutral pH in a wide array of buffers such as WRA, glycine, HEPES, MOPS,Bis-Tris, alanine, acetate or even saline.

6. REFERENCES

The following literature citations as well as those cited above areincorporated in pertinent part by reference herein for the reasons citedin the above text:

Agre and Cartron, “Molecular Biology of the Rh Antigens,” Blood,78:551-563, 1991.

Anderson, “The Experimental Production of Erythroblastosis Foetalis inRabbits,” Brit. J Haemat., 2:44-60, 1956.

Bang et al., “Ultrasound-Guided Fetal Intravenous Transfusion for SevereRhesus Hemolytic Disease,” Brit. Med. J, 284:373-374, 1982.

Berkowitz et al., “Intrauterine Intravascular Transfusions for SevereRed Blood Cell Isoimmunization: Ultrasound-Guided PercutaneousApproach,” Am. J Obstet. Gynecol., 155:574-581, 1986.

Carter, “Preliminary Reporter on a Substance Which Inhibits Anti-RhSerum,” Am. J Clin. Path., 17:646-649, 1947.

Carter, “Rh Hapten: Its Preparation, Assay and Nature,” J. Immunol.,61:79-88, 1949.

Carter, “Preparation and Assay of a Red Cell Fraction of the Rh Factor,”Am. J Clin. Path., 21:561-565, 1951.

Carter, “A Survey of the Rh Hapten,” Texas Reports on Biol. and Med.,17:175-177, 1959.

Carter, “The Use of Rh Hapten in Prevention of Losses Due toErythroblastosis Fetalis,” Proc. 7th Congress European Soc. Haematol.,Part II, 1215-1218, 1960.

Carter, “The Results in Clinical Use of Rh Hapten,” Am. J Ob. Gyn.,102:447-450, 1968.

Carter et al., “Evaluation of Rh Hapten,” Am. J Ob. Gyn., 72:655-659,1956.

Carter and Lewis, “The Effects of Orally Administered Rh Hapten. A Studyof 17 Cases,” Am. J. Ob. Gyn., 76:1286-1287, 1958.

Cohen, “The Immunogenetics of Cellular Antigen Systems of the Rabbit,”In: Oral Immunogenetics and Tissue Transplantation, Eds. G. R. Riviereand W. H. Hildemann, Elsevier North Holland, Amsterdam, p. 183-197,1982.

Cohen and Tissot, “Specialized Research Applications: II SerologicalGenetics,” In: The Biology of the Laboratory Rabbits, Eds. S. H.Weisbroth, R. E. Flatt and A. L. Kraus, Academic Press, New York, p.167-177, 1974.

Dippel, “The Prevention of Erythroblastosis Fetalis by the Use of RhHapten,” Southern Med. J, 45:954-960, 1952.

Ehrenberg, “Report on Treatment of Obstetric Rh Isoimmunization withHapten,” The Journal-Lancet, 75:275-277, 1955.

Garner et al., “Prediction of the Severity of Haemolytic Disease of theNewborn,” Vox. Sang., 68:169-176, 1995.

Goldsmith, “Experiences with Rh Hapten,” Bull. Univ. Minnesota Hospitalsand Minnesota Medical Foundation, 20:188-194, 1948.

Goldsmith, “Experiences with Rh Hapten,” Wisconsin Med. J, May, 1950.

Good and Izawa, Meth. Enzymol., 24(B):53, 1972.

Grannum et al., “In Utero Exchange Transfusion in SevereErythroblastosis Fetalis by Direct Intravascular Transfusion,” New Eng.J Med., 314:1431-1434, 1986.

Iyer et al., “Distribution of IgG Subtypes in Maternal Anti-D Sera andTheir Prognostic Value in Rh Haemolytic Disease of the New-Born,” ActaHaematol., 88::78-81, 1992.

Kellner and Hedal, “Experimental Erythroblastosis Fetalis in Rabbits. I.Characterization of a Pair of Allelic Blood Group Factors and TheirSpecific Immune Isoantibodies,” J. Exp. Med, 97:33-48, 1953.

Liley, “Intrauterine Transfusion of Foetus in Haemolytic Disease,” BritMed. J., 2:1107-1109, 1963.

Moore et al., “Isolation of Membrane Components Associated with HumanRed Cell Antigens Rh(D), (c), (E) and Fya,” Nature, 295:529-531, 1982.

Paradis et al., “Protective Effect of the Membrane Skeleton on theImmunologic Reactivity of the Human Red Cell Rho(D) Antigen,” J.Immunol., 137:240-244, 1986.

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare as described in the claims below.

What is claimed is:
 1. A method of detecting in a sample an antibodyspecific for a antigenically-active antigen, said method comprising: (a)contacting said sample with a protein or peptide comprising an isolatedand purified antienically-active Rh antigen protein or peptide, underconditions effective to allow the formation of an immune complex; and(b) detecting the immune complex so formed.
 2. The method of claim 1,wherein said antibody is an anti-Rh antibody.
 3. The method of claim 2,wherein said antibody is a human or rabbit antibody.
 4. The method ofclaim 3, wherein said antibody is an anti-D antibody, an anti-cantibody, an anti-C antibody, an anti-e antibody, an anti-E antibody, ananti-A antibody, and anti-B antibody, or an anti-F antibody.
 5. Themethod of claim 1, wherein said sample is a blood, serum, plasma,cerebrospinal fluid, lymph, synovial fluid, tissue sample, or culturesupernatant.
 6. The method of claim 1, wherein said protein or peptideis linked to a detectable label.
 7. The antibody of claim 6, whereinsaid protein or peptide is linked to a radioactive label, a fluorogeniclabel, a nuclear magnetic spin resonance label, biotin or an enzyme thatgenerates a colored product upon contact with a chromogenic substrate.8. An immunodetection kit comprising, in suitable container means, aprotein or peptide comprising an isolated and purifiedantigenically-active Rh antigen protein or peptide, and animmunodetection reagent.
 9. The immunodetection kit of claim 8, whereinthe immunodetection reagent is a detectable label that is linked to saidprotein or peptide.
 10. The immunodetection kit of claim 9, wherein theimmunodetection reagent is a detectable label that is linked to a secondantibody that has binding affinity for said protein or peptide.
 11. Theimmunodetection kit of claim 10, wherein the immunodetection reagent isa detectable label that is linked to a second antibody that has bindingaffinity for a human Rh blood group antigen protein or peptide.
 12. Theimmunodetection kit of claim 10, wherein said Rh antigen is a D antigen,a c antigen, a C antigen, an e antigen, an E antigen, an A antigen, a Bantigen, or an F antigen.